Biocatalysts for ezetimibe synthesis

ABSTRACT

The present disclosure relates to non-naturally occurring polypeptides useful for preparing Ezetimibe, polynucleotides encoding the polypeptides, and methods of using the polypeptides.

The present application is a Continuation of co-pending U.S. patentapplication Ser. No. 16/032,319, filed Jul. 11, 2018, which is is aContinuation of U.S. patent application Ser. No. 15/478,466, filed Apr.4, 2017, now U.S. Pat. No. 10,053,673, which is a Continuation of U.S.patent application Ser. No. 15/179,503, filed Jun. 10, 2016, now U.S.Pat. No. 9,644,189, which is a Divisional of U.S. patent applicationSer. No. 14/692,964, filed Apr. 22, 2015, now U.S. Pat. No. 9,388,391,which is a Divisional of U.S. patent application Ser. No. 13/695,856,filed Nov. 2, 2012, now U.S. Pat. No. 9,040,262, which is a nationalstage application filed under 35 USC § 371, and claims priority toPCT/US2011/035194, filed May 4, 2011, and U.S. Prov. Pat. Appln. Ser.No. 61/331,245, filed May 4, 2010, each of which is hereby incorporatedby reference herein.

1. TECHNICAL FIELD

The present disclosure relates to biocatalysts and methods of using thebiocatalysts.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of CX2-037USP1_ST25.txt with a creation date of May 3, 2010, and asize of 281 kilobytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

3. BACKGROUND

The present disclosure relates to improved biocatalysts and improvedbiocatalytic processes for the preparation of the active pharmaceuticalingredient, (1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone) (shown belowas compound (1)) and derivatives and analogs thereof.

Compound (1) is commonly known as Ezetimibe and is the active ingredientin ZETIA®, manufactured by Merck/Schering-Plough Pharmaceuticals.Ezetimibe has been approved by the United States Food and DrugAdministration for use in patients with high cholesterol to reduce LDLcholesterol and total cholesterol (see e.g., U.S. Pat. No. 6,207,822).Ezetimibe lowers high levels of blood cholesterol by selectivelyinhibiting the intestinal absorption of cholesterol and relatedphytosterols. Ezetimibe is commercially available in combination withsimvastatin in the VYTORIN™ formulation from MSP Pharmaceuticals, Inc.

Numerous compounds that are analogs of Ezetimibe and being developed aspossible therapeutics for lowering cholesterol are also known in the art(see e.g., PCT publications WO2006/17257A2, WO 2008/085300A1, andWO2008/039829A2).

Synthetic processes for the production of Ezetimibe and Ezetimibederivatives have been previously disclosed. A variety of publicationshave disclosed chemical synthesis using a late reduction scheme thatdelays the reduction of the alcohol to the carbonyl to the last step ofthe reaction: U.S. Pat. Nos. 5,886,171, 5,738,321, WO 2005/0066120, WO2007/030721, WO 2007/120824, WO 2007/119106, WO 2007/072088, WO2007/030721, and WO 2007/120824.

U.S. Pat. No. 6,133,001 and WO 2000/060107 disclose using certainmicroorganisms (e.g., Rhodococcus fascians ATCC No. 202210 or Geotrichumcandidum ATCC No. 74487) to carry out the stereoselective reduction of1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)propyl)]-4(S)-(4-hydroxyphenyl)-2-azetidinoneto1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-propyl)]-4(S)-(4-hydroxyphenyl)-2-azetidinone.This is a microbial process, however, carried out under whole cellfermentation conditions.

WO 2008/151324A1 discloses using certain commercially availableketoreductase biocatalysts to prepare Ezetimibe and protected Ezetimibeanalogs from the corresponding precursor ketone compounds. Thebiocatalysts and processes disclosed therein, however, use low substrateloadings (25 g/L or less), a GDH/glucose cofactor regeneration system,and result in low percentage conversion of substrate to the Ezetimibeproduct (˜65% yield).

US20100062499A1 discloses engineered ketoreductase enzymes, and methodsof using the engineered ketoreductase enzymes to convert the diketonecompound,5-(4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,-5-dione,to the chiral alcohol,(4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolid-in-2-one.This chiral alcohol made biocatalytically is an early stage intermediatethat can be used in a process for making Ezetimibe.

It is desirable to have improved biocatalysts and a biocatalytic processhaving increased efficiency for use in a late stage biocatalyticreduction scheme for preparing Ezetimibe in high diastereomeric excess(>98% d.e.). Particularly desirable would be engineered biocatalystscapable of increased activity in large scale processes having highsubstrate loadings (e.g., >50 g/L), high percent conversion (e.g., >90%in 24 h), without the need for an additional cofactor regeneratingenzyme, and capable of yielding Ezetimibe as product in high purity anddiastereomeric excess.

4. SUMMARY

The present disclosure provides non-naturally occurring polypeptideshaving ketoreductase activity, polynucleotides encoding thepolypeptides, methods of the making the polypeptides, and methods ofusing the polypeptides for the biocatalytic conversion of theketo-phenol substrate,1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone(compound (2) below) to the chiral (S)-alcohol product,1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone(i.e., compound (1) commonly referred to as Ezetimibe) as shown inScheme 1.

While naturally occurring ketoreductase polypeptides do not efficientlyconvert compound (2) to compound (1), the non-naturally occurring,engineered, ketoreductase polypeptides of the present disclosure arecapable of carrying out this conversion with improved propertiesincluding, high diastereomeric excess (e.g., at least about 99% d.e.),increased activity (e.g., at least about 10-fold increased activityrelative to the reference polypeptide SEQ ID NO:2), high percentconversion (e.g., at least about 90% conversion in 24 h), in thepresence of high substrate loadings (e.g., at least about 50 g/Lcompound (2)), and without any cofactor regenerating enzyme other thanthe engineered ketoreductase polypeptide.

The non-naturally occurring polypeptides of the present disclosurecapable of converting compound (2) to compound (1) with at least 2-fold,at least 10-fold, at least 25-fold, at least 40-fold, or at least60-fold increased activity relative to the activity of the referencepolypeptide of SEQ ID NO: 2, are synthetic variants of the naturallyoccurring ketoreductase of Lactobacillus kefir, and comprise amino acidsequences that have one or more residue differences as compared to thereference sequence of the synthetic variant ketoreductase polypeptide ofSEQ ID NO:2. The residue differences occur at residue positions thataffect functional properties of the enzyme including activity (e.g.,percent conversion of substrate to product), stereoselectivity,substrate and/or product binding (e.g., resistance to substrate and/orproduct inhibition), thermostability, solvent stability, expression, orvarious combinations thereof. Accordingly, in some embodiments, thepolypeptides of the disclosure can have one or more residue differencesas compared to SEQ ID NO:2 at the following residue positions: X21, X25,X40, X64, X93, X94, X95, X96, X99, X108, X117, X127, X147, X148, X150,X152, X153, X155, X190, X195, X196, X201, X202, X203, X204, X205, X206,X207, X211, X221, X223, and X226 Amino acid residues that can be presentat these positions are described in detail in the descriptions herein.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide of capable of converting compound (2) to compound(1) comprising an amino acid sequence selected from any one of SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, or 168.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, and comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, and furthercomprises a set of amino acid residue differences as compared to SEQ IDNO:2 of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168. In some embodiments, inaddition to the set of amino acid residue differences of any one of thenon-naturally occurring polypeptides of SEQ ID NO: 4 through SEQ ID NO:168, the sequence of the non-naturally occurring polypeptide can furthercomprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residuedifferences at other amino acid residue positions as compared to the SEQID NO: 2. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to SEQ ID NO: 2.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, which comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 and atleast the following features: residue at position corresponding to X40is R; residue at position corresponding to X153 is I, or L; residue atposition corresponding to X190 is A or P; residue at positioncorresponding to X196 is T; residue at position corresponding to X199 isF, or W; and residue at position corresponding to X206 is I. In someembodiments, the amino acid sequence further comprises at least onefeature or group of features selected from: (a) residue at position X93is A and residue at position X94 is T; (b) residue at position X93 is Aand residue at position X94 is S; (c) residue at position X93 is A andresidue at position X94 is S; (d) residue at position X93 is I andresidue at position X94 is S; (e) residue at position X203 is G; (f)residue at position X202 is G and residue at position X203 is G; or (f)residue at position X201 is A, residue at position X202 is G, andresidue at position X203 is G.

In some embodiments, any of the non-naturally occurring polypeptides ofthe present disclosure capable of converting compound (2) to compound(1) with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2 and an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 2, can further one or morefeatures selected from: residue at position corresponding to X21 is R orF; residue at position corresponding to X25 is R, T, or N; residue atposition corresponding to X40 is R; residue at position corresponding toX64 is V; residue at position corresponding to X93 is A; residue atposition corresponding to X94 is T, S, or P; residue at positioncorresponding to X95 is V, or M; residue at position corresponding toX96 is V, G, A, N, S, P, or T; residue at position corresponding to X99is L; residue at position corresponding to X108 is H; residue atposition corresponding to X117 is A, or G; residue at positioncorresponding to X127 is K, or Q; residue at position corresponding toX147 is M, or I; residue at position corresponding to X148 is I; residueat position corresponding to X150 is H, or A; residue at positioncorresponding to X152 is N, or F; residue at position corresponding toX153 is I, or L; residue at position corresponding to X155 is C; residueat position corresponding to X190 is A; residue at positioncorresponding to X195 is M; residue at position corresponding to X196 isT, A, S, C, or N; residue at position corresponding to X199 is F, or W;residue at position corresponding to X201 is I, L, or A; residue atposition corresponding to X202 is L, N, V, or G; residue at positioncorresponding to X203 is G; residue at position corresponding to X204 isV, or A; residue at position corresponding to X205 is V; residue atposition corresponding to X206 is I; residue at position correspondingto X207 is T, C, I, or N; residue at position corresponding to X211 isK; residue at position corresponding to X221 is D; residue at positioncorresponding to X223 is I; residue at position corresponding to X226 isV.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise a sequence having various combinationsof the residue differences as compared to SEQ ID NO:2 disclosed hereinat residue positions affecting enzymatic activity, thermostability,solvent stability, and cofactor binding.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise a sequence having one or more amino acidresidue differences as compared to SEQ ID NO: 2 at residue positionsaffecting activity for conversion of compound (2) to compound (1), areselected from the following position: X21; X25; X64; X93; X94; X95; X96;X99; X108; X117; X127; X147; X148; X150; X152; X153; X155; X163; X190;X195; X196; X199; X201; X202; X203; X204; X205; X206; X207; X211; X221;X223; and X226. In some embodiments, specific amino acid differences atresidue positions resulting in increased activity for conversion ofcompound (2) to compound (1) relative to the reference polypeptide ofSEQ ID NO: 2 can be selected from the following substitutions: L21FR;D25NRT; A64V; I93AT; A94PST; L95MV; Q96ANGPSTV; V99L; R108DHK; S117AG;R127KQ; L147IM; V148I; D150H; M152NF; V153IL; A155C; V163I; C190A;L195M; V196ACNST; D199FW; G201AIL; A202GLNV; E203G; E204AV; M205V;M206I; S207TCIN; R211K; N221D; V223I; and I226V.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise an amino acid sequence having one ormore residue differences as compared to SEQ ID NO: 2 at residuepositions affecting thermostability, which positions include thefollowing: X21; X93; X94; X117; X127; X147; X195; and X199. In someembodiments, specific amino acid differences at residue positionsresulting in increased thermostability relative to the referencepolypeptide of SEQ ID NO: 2 can be selected from the followingsubstitutions: L21F; A93T; S94A; S117GA; R127K; L147I; L195M; and D199W.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise an amino acid sequence having residuedifferences as compared to SEQ ID NO: 2 at residue positions affectingsolvent stability, which positions include X25; X147; and X221. In someembodiments, specific amino acid differences at residue positionsresulting in increased solvent stability relative to the referencepolypeptide of SEQ ID NO: 2 can be selected from the followingsubstitutions: D25R; L147M; and N221D.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise an amino acid sequence having residuedifferences as compared to SEQ ID NO: 2 at residue positions affectingcofactor binding, which positions include X40. In some embodiments,specific amino acid differences at residue positions affecting cofactorbinding can be selected from the following substitutions: H40R.

In addition to the residue position specified above, various otherresidue differences relative to SEQ ID NO:2 can be present at otherresidue positions in the ketoreductase polypeptides disclosed herein.These can be conservative or non-conservative differences, includingconservative substitutions and non-conservative substitutions. Guidanceon the choice of amino acid residues at the specified positions isprovided in the detailed description.

In some embodiments, the present disclosure provides polynucleotidesencoding the non-naturally occurring polypeptides capable of convertingcompound (2) to compound (1), as well as expression vectors comprisingthe polynucleotides, and host cells capable of expressing thepolynucleotides encoding the non-naturally occurring polypeptides.Accordingly, in some embodiments, the present disclosure also providesmethods of manufacturing the non-naturally occurring polypeptidescapable of converting compound (2) to compound (1), wherein the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the non-naturally occurring polypeptide and isolating thepolypeptide from the host cell.

In some embodiments, any of the non-naturally occurring polypeptides ofthe present disclosure can be used in improved processes for carryingout the conversion of compound (2) to compound (1) due to their improvedenzymatic properties including, high diastereomeric excess (e.g., atleast about 99% d.e.), increased activity (e.g., at least about 10-foldincreased activity relative to SEQ ID NO:2), high percent conversion(e.g., at least about 90% conversion in 24 h), in the presence of highsubstrate loadings (e.g., at least about 50 g/L compound (2)), andwithout any cofactor regenerating enzyme other than the non-naturallyoccurring ketoreductase polypeptide. Accordingly, in some embodiments,the present disclosure provides methods using the non-naturallyoccurring polypeptides for preparing compound (1) in diastereomericexcess, wherein the methods comprise: contacting compound (2) with annon-naturally occurring or engineered polypeptide of the presentdisclosure (e.g., as described in Table 2 and elsewhere herein) in thepresence of NADPH or NADH cofactor under suitable reaction conditions.Suitable reactions conditions for the conversion of compound (2) tocompound (1) using the engineered polypeptides of the present disclosureare described in greater detail below, including but not limited toranges of pH, temperature, buffer, solvent system, substrate loading,polypeptide loading, cofactor loading, atmosphere, and reaction time.

In some embodiments, the improved enzymatic activity of the engineeredpolypeptides in the conversion of compound (2) to compound (1) providesfor methods wherein a higher percentage conversion can be achieved witha lower concentration of polypeptide. The use of lower concentration ofthe engineered polypeptide in a method comprising a conversion ofcompound (2) to compound (1) also reduces the amount of residual proteinthat may need to be removed in subsequent steps for purification ofcompound (1). Accordingly, in some embodiments, the methods forpreparing compound (1) of the present disclosure can be carried outwherein the suitable reaction conditions can comprise e.g., a compound(2) substrate loading of at least about 20 g/L, about 40 g/L, about 50g/L, about 75 g/L, about 100 g/L, about 200 g/L, about 250 g/L, about300 g/L, or about 400 g/L; and/or a non-naturally occurring polypeptideconcentration of about 0.1-3.0 g/L, about 0.5-2.75 g/L, about 1.0-2.5g/L, about 1.5-2.5 g/L, about 3 g/L, about 2 g/L, about 1.5 g/L, about1.0 g/L, about 0.75 g/L, or even lower concentration.

In some embodiments, the present disclosure also provides methods forpreparing compound (1) or an analog of compound (1), wherein the methodscomprise contacting compound (2) or an analog of compound (2) with anon-naturally occurring or engineered polypeptide in the presence ofNADPH or NADH cofactor under suitable reaction conditions and furthercomprises chemical steps of product work-up, extraction, isolation,purification, and/or crystallization of compound (1), each of which canbe carried out under a range of conditions disclosed herein.

In some embodiments, the methods for preparing compound (1) using anon-naturally occurring polypeptide of the present disclosure furthercomprise a cofactor recycling system capable of converting NADP⁺ toNADPH, or NAD⁺ to NADH. The cofactor recycling system can comprise adehydrogenase enzyme (e.g., glucose dehydrogenase, glucose-phosphatedehydrogenase, formate dehydrogenase, or a ketoreductase/alcoholdehydrogenase) and a corresponding substrate (e.g., glucose,glucose-6-phosphate, formate, or secondary alcohol). In someembodiments, the co-factor recycling system comprises a ketoreductasepolypeptide and a secondary alcohol, preferably isopropanol. In someembodiments of the methods of the present disclosure, the non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)is also capable of converting a secondary alcohol (e.g., isopropanol) toits corresponding secondary ketone (e.g., acetone), and the method ofpreparing compound (1) further comprises a co-factor recycling systemcomprising the non-naturally occurring polypeptide and a secondaryalcohol.

In some embodiments, an analog of compound (1) can be prepared indiastereomeric excess from an analog of compound (2) using the abovedescribed methods. In some embodiments, the analog of compound (1)prepared using the methods comprises a compound of Formula Ia:

wherein,

X is C or S;

R¹ is selected from —H, —F, —Cl, —Br, or —I;

R² is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle;

R³ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle;

R⁴ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group); and

R⁵ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle.

Accordingly, in some embodiments the present disclosure provides amethod of preparing a compound of Formula Ia in diastereomeric excesscomprising: contacting a compound of Formula IIa

wherein, X, R¹, R², R³, R⁴, and R⁵, are defined as above for Formula Ia,with an engineered polypeptide of the present disclosure (e.g., asdescribed in Table 2 and elsewhere herein) in the presence of NADPH orNADH cofactor under suitable reaction conditions.

5. DETAILED DESCRIPTION

The present disclosure provides highly stereoselective and efficientbiocatalysts capable of mediating the conversion of1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinoneto1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinonein diastereomeric excess. The biocatalysts described herein have beendesigned by changing the amino acid sequence of a naturally occurringketoreductase to form polypeptides with the desired enzymaticproperties, e.g., enzyme activity, stereoselectivity, by productformation, thermostability, and expression. The detailed descriptionthat follow describes the polypeptides and processes for carrying outthe conversion of1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinoneto1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone in diastereomeric excess.

For the descriptions herein and the appended claims, the singular forms“a”, “an” and “the” include plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a polypeptide”includes more than one polypeptide, and reference to “a compound” refersto more than one compound.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure.

5.2 Definitions

The technical and scientific terms used in the descriptions herein willhave the meanings commonly understood by one of ordinary skill in theart, unless specifically defined otherwise. Accordingly, the followingterms are intended to have the following meanings.

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Non-naturally occurring” or “engineered” or “recombinant” when used inthe present disclosure with reference to, e.g., a cell, nucleic acid, orpolypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Numerous other algorithms are available that function similarly to BLASTin providing percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which anothersequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity. The term “reference sequence” is not intended to belimited to wild-type sequences, and can include engineered or alteredsequences. For example, in some embodiments, a “reference sequence” canbe a previously engineered or altered amino acid sequence.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredketoreductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as stereoselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereo selectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate (e.g., compound (2),1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone)to its corresponding product (e.g., compound (1),1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone)with at least about 85% stereoisomeric excess.

“Increased enzymatic activity” or “increased activity” refers to animproved property of an engineered enzyme, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount ofketoreductase) as compared to a reference enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. The ketoreductase activity can bemeasured by any one of standard assays used for measuringketoreductases, such as change in substrate or product concentration, orchange in concentration of the cofactor (in absence of a cofactorregenerating system). Comparisons of enzyme activities are made using adefined preparation of enzyme, a defined assay under a set condition,and one or more defined substrates, as further described in detailherein. Generally, when enzymes in cell lysates are compared, thenumbers of cells and the amount of protein assayed are determined aswell as use of identical expression systems and identical host cells tominimize variations in amount of enzyme produced by the host cells andpresent in the lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “enzymatic activity”or “activity” of a ketoreductase polypeptide can be expressed as“percent conversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed to a set oftemperature conditions (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (e.g., more than 60% to 80% for example)after exposure to elevated temperatures.

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln(Q), Asp (D), Lys (K) and Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include Glu (E) and Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include Arg (R) and Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include Asn (N), Gln (Q), Ser (S)and Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include Pro (P), Ile (I), Phe (F), Val (V), Leu (L), Trp(W), Met (M), Ala (A) and Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include Phe (F), Tyr (Y) and Trp (W). Although owing to the itsheteroaromatic ring side chain His (H) is classified as an aromaticresidue, it may also be classified as a basic residue owing to pKa ofits heteroaromatic nitrogen atom.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeGly (G), Leu (L), Val (V), Ile (I), Met (M) and Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid Cys (C) is unique in that it can form disulfide bridgeswith other Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The ability of Cys (and other aminoacids with —SH containing side chains) to exist in a polypeptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether it contributes net hydrophobic or hydrophilic character to thepolypeptide. While Cys exhibits a hydrophobicity of 0.29 according tothe normalized consensus scale of Eisenberg (Eisenberg et al., 1984,supra), it is to be understood that for purposes of the presentdisclosure, Cys is classified into its own unique group.

The amino acid Pro (P) has a conformationally constrained nature.Although it has hydrophobic properties, as used herein, Pro (P) or othersimilar residues is classified as a “conformationally constrained.”

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acid orresidue containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include Ser (S) and Thr (T). While L-Tyr(Y) contains a hydroxyl moiety, it is classified herein as an aromaticamino acid or residue.

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition X3, where the reference sequence has a glutamine, refers to achange of the residue at position X3 to any residue other thanglutamine. As disclosed herein, an enzyme can include one or moreresidue differences relative to a reference sequence, where multipleresidue differences typically are indicated by a list of the specifiedpositions where changes are made relative to the reference sequence. Theresidue differences can be non-conservative changes or conservativechanges. In some embodiments, the residue differences can beconservative substitutions, non-conservative substitutions, or acombination of non-conservative and conservative substitutions. For thedescriptions of the non-naturally occurring polypeptides herein, theamino acid residue position in the reference sequence is determined inthe ketoreductase polypeptide beginning from the initiating methionine(M) residue (i.e., M represents residue position 1), although it will beunderstood by the skilled artisan that this initiating methionineresidue may be removed by biological processing machinery, such as in ahost cell or in vitro translation system, to generate a mature proteinlacking the initiating methionine residue. The polypeptide sequenceposition at which a particular amino acid or amino acid change (“residuedifference”) is present is sometimes described herein as “Xn”, or“position n”, where n refers to the residue position with respect to thereference sequence. Where applicable, a specific substitution mutation,which is a replacement of the specific residue in a reference sequencewith a different specified residue may be denoted by the conventionalnotation “X(number)Y”, where X is the single letter identifier of theresidue in the reference sequence, “number” is the residue position inthe reference sequence, and Y is the single letter identifier of theresidue substitution in the engineered sequence.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains, and thus typically involvessubstitution of the amino acid in the polypeptide with amino acidswithin the same or similar defined class of amino acids. By way ofexample and not limitation, an amino acid with an aliphatic side chainmay be substituted with another aliphatic amino acid, e.g., alanine,valine, leucine, and isoleucine; an amino acid with hydroxyl side chainis substituted with another amino acid with a hydroxyl side chain, e.g.,serine and threonine; an amino acids having aromatic side chains issubstituted with another amino acid having an aromatic side chain, e.g.,phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with abasic side chain is substituted with another amino acid with a basisside chain, e.g., lysine and arginine; an amino acid with an acidic sidechain is substituted with another amino acid with an acidic side chain,e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are provided inTable 1.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification of the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the polypeptidewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered ketoreductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification of the polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the improved engineered ketoreductase enzymes comprise insertions of oneor more amino acids to the naturally occurring ketoreductase polypeptideas well as insertions of one or more amino acids to other improvedketoreductase polypeptides. Insertions can be in the internal portionsof the polypeptide, or to the carboxy or amino terminus. Insertions asused herein include fusion proteins as is known in the art. Theinsertion can be a contiguous segment of amino acids or separated by oneor more of the amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, 90%, 95%, 98%, and 99%of the full-length ketoreductase polypeptide, for example thepolypeptide of SEQ ID NO:2.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved ketoreductase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theengineered ketoreductase polypeptides of the present disclosure can bean isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure engineered ketoreductasepolypeptide composition will comprise about 60% or more, about 70% ormore, about 80% or more, about 90% or more, about 95% or more, and about98% or more of all macromolecular species by mole or % weight present inthe composition. Solvent species, small molecules (<500 Daltons), andelemental ion species are not considered macromolecular species. In someembodiments, the isolated improved ketoreductase polypeptide is asubstantially pure polypeptide composition.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. In some embodiments, the polynucleotides encoding theketoreductase enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the polynucleotide of interest. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Cofactor regeneration system” refers to a set of reactants thatparticipate in a reaction that reduces the oxidized form of the cofactor(e.g., NADP+ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD+ or NADP+, respectively, are known in the art and may be usedin the methods described herein.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of D-glucose and NAD⁺ or NADP to gluconic acid and NADH orNADPH, respectively.

The term “secondary alcohol dehydrogenase” is used herein to refer to anNAD⁺ or NADP⁺-dependent enzyme that catalyzes the conversion of asecondary alcohol (e.g., isopropanol) and NAD⁺ or NADP⁺ to a ketone andNADH or NADPH, respectively.

“Alkyl” refers to groups of from 1 to 12 carbon atoms inclusively,either straight chained or branched, more preferably from 1 to 8 carbonatoms inclusively, and most preferably 1 to 6 carbon atoms inclusively.

“Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one double bond butoptionally containing more than one double bond.

“Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one triple bond butoptionally containing more than one triple bond, and additionallyoptionally containing one or more double bonded moieties.

“Alkoxy” refers to the group alkyl-O— wherein the alkyl group is asdefined above including optionally substituted alkyl groups as alsodefined above.

“Alkenoxy” refers to the group alkenyl-O— wherein the alkenyl group isas defined above including optionally substituted alkenyl groups as alsodefined above.

“Alkynoxy” refers to the group alkynyl-O— wherein the alkynyl group isas defined above including optionally substituted alkynyl groups as alsodefined above.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to14 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Preferred arylsinclude phenyl, naphthyl and the like.

“Arylalkyl” refers to aryl -alkyl- groups preferably having from 1 to 6carbon atoms inclusively in the alkyl moiety and from 6 to 10 carbonatoms inclusively in the aryl moiety. Such arylalkyl groups areexemplified by benzyl, phenethyl and the like.

“Arylalkenyl” refers to aryl -alkenyl- groups preferably having from 2to 6 carbon atoms in the alkenyl moiety and from 6 to 10 carbon atomsinclusively in the aryl moiety.

“Arylalkynyl” refers to aryl -alkynyl- groups preferably having from 2to 6 carbon atoms inclusively in the alkynyl moiety and from 6 to 10carbon atoms inclusively in the aryl moiety.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups. suchcycloalkyl groups include, by way of example, single ring structuressuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures such as adamantyl, and the like.

“Cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 12 carbonatoms inclusively having a single cyclic ring or multiple condensedrings and at least one point of internal unsaturation, which can beoptionally substituted with from 1 to 3 alkyl groups. examples ofsuitable cycloalkenyl groups include, for instance, cyclobut-2-enyl,cyclopent-3-enyl, cyclooct-3-enyl and the like.

“Cycloalkylalkyl” refers to cycloalkyl -alkyl- groups preferably havingfrom 1 to 6 carbon atoms inclusively in the alkyl moiety and from 6 to10 carbon atoms inclusively in the cycloalkyl moiety. Suchcycloalkylalkyl groups are exemplified by cyclopropylmethyl,cyclohexylethyl and the like.

“Cycloalkylalkenyl” refers to cycloalkyl -alkenyl- groups preferablyhaving from 2 to 6 carbon atoms inclusively in the alkenyl moiety andfrom 6 to 10 carbon atoms inclusively in the cycloalkyl moiety. Suchcycloalkylalkenyl groups are exemplified by cyclohexylethenyl and thelike.

“Cycloalkylalkynyl” refers to cycloalkyl -alkynyl- groups preferablyhaving from 2 to 6 carbon atoms inclusively in the alkynyl moiety andfrom 6 to 10 carbon atoms inclusively in the cycloalkyl moiety. Suchcycloalkylalkynyl groups are exemplified by cyclopropylethynyl and thelike.

“Heteroaryl” refers to a monovalent aromatic heterocyclic group of from1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to heteroaryl -alkyl- groups preferably havingfrom 1 to 6 carbon atoms inclusively in the alkyl moiety and from 6 to10 atoms inclusively in the heteroaryl moiety. Such heteroarylalkylgroups are exemplified by pyridylmethyl and the like.

“Heteroarylalkenyl” refers to heteroaryl -alkenyl- groups preferablyhaving from 2 to 6 carbon atoms inclusively in the alkenyl moiety andfrom 6 to 10 atoms inclusively in the heteroaryl moiety.

“Heteroarylalkynyl” refers to heteroaryl -alkynyl- groups preferablyhaving from 2 to 6 carbon atoms inclusively in the alkynyl moiety andfrom 6 to 10 atoms inclusively in the heteroaryl moiety.

“Heterocycle” refers to a saturated or unsaturated group having a singlering or multiple condensed rings, from 1 to 8 carbon atoms inclusivelyand from 1 to 4 hetero atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Preferredheterocycles include piperidinyl, pyrrolidinyl and tetrahydrofuryl.

Examples of heterocycles and heteroaryls include, but are not limitedto, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,pyrrolidine, indoline and the like.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable.

The term “suitable reaction conditions” refers to those conditions inthe biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, T, pH, buffers, co-solvents, etc.)under which a non-naturally occurring ketoreductase polypeptide of thepresent disclosure is capable of converting compound (2) to compound (1)(or compound of Formula IIa to compound of Formula Ia). Exemplary“suitable reaction conditions” are provided in the present disclosureand illustrated by the Examples.

5.3 Non-Naturally Occurring or Engineered Ketoreductase Polypeptides

Enzymes belonging to the ketoreductase (KRED) or carbonyl reductaseclass (EC1.1.1.184) are useful for the synthesis of optically activealcohols from the corresponding prostereoisomeric ketone substrates andby stereospecific reduction of corresponding racemic aldehyde and ketonesubstrates. KREDs typically convert a ketone or aldehyde substrate tothe corresponding alcohol product, but may also catalyze the reversereaction, oxidation of an alcohol substrate to the correspondingketone/aldehyde product. The reduction of ketones and aldehydes and theoxidation of alcohols by enzymes such as KRED requires a co-factor, mostcommonly reduced nicotinamide adenine dinucleotide (NADH) or reducednicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamideadenine dinucleotide (NAD) or nicotinamide adenine dinucleotidephosphate (NADP+) for the oxidation reaction. NADH and NADPH serve aselectron donors, while NAD and NADP serve as electron acceptors. It isfrequently observed that KREDs and other alcohol dehydrogenases accepteither the phosphorylated or the non-phosphorylated co-factor (in itsoxidized and reduced state).

KREDs are being used increasingly in place of chemical procedures forthe conversion of different keto and aldehyde compounds to chiralalcohol products. These biocatalytic conversions can employ whole cellsexpressing the ketoreductase for biocatalytic ketone reductions, orpurified enzymes, particularly in those instances where presence ofmultiple ketoreductases in whole cells would adversely affect theenantiomeric purity and yield of the desired product. For in vitroapplications, a co-factor (NADH or NADPH) regenerating enzyme such asglucose dehydrogenase (GDH), formate dehydrogenase etc. is used inconjunction with the ketoreductase.

Examples illustrating the use of naturally occurring or engineered KREDsin biocatalytic processes to generate useful chemical compounds includeasymmetric reduction of 4-chloroacetoacetate esters (Zhou, J. Am. Chem.Soc. 1983 105:5925-5926; Santaniello, J. Chem. Res. (S) 1984:132-133;U.S. Pat. Nos. 5,559,030; 5,700,670 and 5,891,685), reduction ofdioxocarboxylic acids (e.g., U.S. Pat. No. 6,399,339), reduction oftert-butyl (S)-chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No.6,645,746 and WO 01/40450), reduction pyrrolotriazine-based compounds(e.g., U.S. application No. 2006/0286646); reduction of substitutedacetophenones (e.g., U.S. Pat. No. 6,800,477); and reduction ofketothiolanes (WO 2005/054491).

KREDs can be found in a wide range of bacteria and yeasts (for reviews:Kraus and Waldman, Enzyme catalysis in organic synthesis Vols. 1&2.VCHWeinheim 1995; Faber, K., Biotransformations in organic chemistry, 4thEd. Springer, Berlin Heidelberg New York. 2000; Hummel and Kula Eur. J.Biochem. 1989 184:1-13). Several KRED gene and enzyme sequences havebeen reported, including: Candida magnoliae (Genbank Acc. No. JC7338;GI:11360538); Candida parapsilosis (Genbank Acc. No. BAA24528.1;GI:2815409), Sporobolomyces salmonicolor (Genbank Acc. No. AF160799;GI:6539734); Lactobacillus kefir (Genbank Acc. No. AAP94029.1; GI:33112056); Lactobacillus brevis (Genbank Acc. No. 1NXQ_A; GI: 30749782);and Thermoanaerobium brockii (Genbank Acc. No. P14941; GI: 1771790).

These naturally occurring ketoreductase polypeptides do not efficientlyconvert compound (2) to compound (1). The non-naturally occurring,engineered, ketoreductase polypeptides of the present disclosure,however, are capable of carrying out this conversion with improvedproperties including, high diastereomeric excess (e.g., at least about99% d.e.), increased activity (e.g., at least about 10-fold increasedactivity relative to the reference polypeptide SEQ ID NO:2), highpercent conversion (e.g., at least about 90% conversion in 24 h), in thepresence of high substrate loadings (e.g., at least about 50 g/Lcompound (2)), and without any cofactor regenerating enzyme other thanthe engineered ketoreductase polypeptide.

The non-naturally occurring polypeptides of the present disclosure aresynthetic variants of the naturally occurring ketoreductase ofLactobacillus kefir, and comprise amino acid sequences that have one ormore residue differences as compared to the reference sequence of thesynthetic variant ketoreductase polypeptide of SEQ ID NO:2. The residuedifferences occur at residue positions that affect enzyme activity,stereoselectivity, thermostability, expression, or various combinationsthereof. The residue differences provide structural changes that allowthe engineered polypeptides to convert the ketophenol substrate,1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3-oxopropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone(compound (2); MW 407.41) to the chiral alcohol product,1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone(compound (1); MW 409.43) (as illustrated in Scheme 1) with at least2-fold, at least 10-fold, at least 25-fold, at least 40-fold, or atleast 60-fold increased activity relative to the activity of thereference polypeptide of SEQ ID NO: 2. Further these engineeredpolypeptides are capable of highly stereoselective conversion ofcompound (2) to compound (1) in at least about 97%, about 98%, or atleast about 99% diastereomeric excess. Further, in some embodimentsthese non-naturally occurring polypeptides are capable of catalyzing theconversion of compound (2) to compound (1) using added cofactor (NADPHor NADH), or in presence of a co-factor recycling system, for example anappropriate dehydrogenase (e.g., glucose dehydrogenase, formatedehydrogenase or ketoreductase/alcohol dehydrogenase) and a suitabledehydrogenase substrate, such as glucose, glucose-6-phosphate, formate,or a secondary alcohol, e.g., isopropanol. In some embodiments, thenon-naturally occurring ketoreductase polypeptides can function not onlyto convert compound (2) to compound (1), but also function as thesecondary alcohol dehydrogenase of a cofactor recycling system andthereby recycle the cofactor in the presence of a secondary alcohol.Thus, the engineered biocatalysts present disclosure are capable ofproviding highly efficient biocatalytic processes for preparingEzetimibe as substantially enantiomerically pure preparations.

Structure and function information for exemplary non-naturally occurring(or engineered) ketoreductase polypeptides of the present disclosure areshown below in Table 2. The odd numbered sequence identifiers (i.e., SEQID NOs) refer to the nucleotide sequence encoding the amino acidsequence provided by the even numbered SEQ ID NOs, and the sequences areprovided in the electronic sequence listing file accompanying thisdisclosure, which is hereby incorporated by reference herein. The aminoacid residue differences are based on comparison to the referencesequence of SEQ ID NO: 2, which is an engineered ketoreductase havingthe following 19 residue differences relative to the amino acid sequenceof the naturally occurring wild-type ketoreductase of Lactobacilluskefir (Genbank acc. No. AAP94029.1; GI: 33112056): D3N, G7S, L17Q, V95L,S96Q, G117S, Q127R, E145S, F147L, T152M, L153V, L176V, Y190C, D198K,L199D, E200P, K211R, I223V, and A241S. The activity of each engineeredpolypeptide relative to the reference polypeptide of SEQ ID NO: 2 wasdetermined as conversion of substrate of compound (2) to product ofcompound (1) over a 24 h period at room temperature in a 96-well plateformat assay of cell lysates containing the engineered polypeptides.General assay protocol and reaction conditions were as follows (withexceptions noted in Table 2): 60 μL of a 13.33 g/L solution of thecompound (2) in toluene:IPA:acetone (v/v/v ratio of 5:9:1) added to eachwell of a Costar™ deep-well 96-well plate; subsequently, 120 μL of a 0.8g/L solution of NADP in 100 mM TEA buffer, pH 7.0 containing 1 mM MgSO₄added to each well; finally, 20 μL of a freshly prepared suspension oflysed cells (i.e., cells expressing the variant polypeptide) in lysisbuffer were added to make the total volume in each well 200 μL. Finalconditions in each well (except as noted below): [compound (2)]=4 g/L,[NADP]=0.5 g/L, Solvent=toluene:IPA:acetone:buffer (relative % volumesof 10:18:2:70). The plate was then heat sealed and shaken for 24 h at RT(or 37° C.) before 0.8 mL of acetonitrile was added to each well toquench the reaction. The levels of activity (i.e., “+” “++” “+++” etc.)are defined as follows: “+” indicates at least equal to but less than 2times the activity of SEQ ID NO: 2; “++” indicates at least 2 times butless than 10 times the activity of SEQ ID NO: 2; “+++” indicates atleast 10 times but less than 25 times the activity of SEQ ID NO: 2;“++++” indicates at least 25 times but less than 40 times the activityof SEQ ID NO: 2; “+++++” indicates at least 40 times but less than 60times the activity of SEQ ID NO: 2; “++++++” indicates at least 60 timesthe activity of SEQ ID NO: 2.

TABLE 2 Activity SEQ (relative ID to NO Residue differences SEQ ID(nt/aa) (relative to SEQ ID NO:2) NO: 2)   3/4 H40R; V153I; C190A;E204V; ++   5/6 H40R; I93A; A94T; V153I; C190A; ++ V196T; D199F; M206I;  7/8 H40R; +   9/10 H40R; V148I; +  11/12 H40R; V196A; +  13/14 H40R;S207T; ++  15/16 H40R; Q96V; +  17/18 H40R; V196S; +  19/20 H40R; V196C;++  21/22 H40R; V196N; ++  23/24 H40R; A202L; ++  25/26 H40R; A202N; + 27/28 H40R; A202V; ++  29/30 H40R; I93A; A94P; V153I; ++ C190A; V196T;D199F; M206I;  31/32 H40R; Q96G; V153I; C190A; ++ V196T; D199F; M206I; 33/34 H40R; Q96A; V153I; C190A; ++ V196T; D199F; M206I;  35/36 H40R;I93A; A94T; V153I; C190A; ++ V196T; D199F; E203G; M206I;  37/38 H40R;I93A; A94T; Q96V; V153I; + C190A; V196T; D199F; M206I;  39/40 H40R;I93A; A94T; V153I; C190A; ++ L195M; V196T; D199F; M206I;  41/42 H40R;I93A; A94T; V153I; C190A; ++ V196T; D199F; M206I; S207C;  43/44 H40R;I93A; A94T; V153I; C190A; ++ V196T; D199F; M206I; S207I;  45/46 H40R;I93A; A94T; V153I; C190A; ++ V196T; D199F; M206I; S207N;  47/48 L21R;D25R; H40R; I93A; A94T; +++¹ S117G; R127K; L147M; V153I;   C190A; V196T;D199F; E203G; M206I; N221D;  49/50 L21F; D25R; H40R; I93A; A94T; +++¹S117G; L147M; V153I; C190A;   V196T; D199F; E203G; M206I; N221D;  51/52H40R; I93A; A94S; Q96N; V153L; +++¹ C190A; V196T; D199F; A202G;   E203G;M206I;  53/54 D25T; H40R; I93A; A94T; R108H; +++¹ V153I; C190A; V196T;D199F;   E203G; M206I;  55/56 H40R; I93A; A94T; D150H; V153I; +++¹C190A; V196T; D199F; E203G;   M206I;  57/58 H40R; I93A; A94T; V153I;C190P; +++¹ V196T; D199F; E203G; M206I;  59/60 H40R; I93A; A94T; V153I;C190A; +++¹ V196T; D199F; E203G; E204A;   M206I;  61/62 H40R; I93A;A94T; V153I; C190A; +++¹ V196T; D199F; E203G; E204V;   M206I;  63/64H40R; I93A; A94T; V153I; C190A; +++¹ V196T; D199F; E203G; M205V;  M206I;  65/66 H40R; I93A; A94T; M152N; V153I; +++¹ C190A; V196T; D199F;E203G;   M206I;  67/68 H40R; I93A; A94T; M152F; V153I; +++¹ C190A;V196T; D199F; E203G;   M206I;  69/70 H40R; I93A; A94T; Q96A; V153I; +++¹C190A; V196T; D199F; E203G;   M206I; S207T;  71/72 D25N; H40R; I93A;A94T; V153I; +++¹ C190A; V196T; D199F; E203G;   M206I;  73/74 D25T;H40R; I93A; A94T; V153I; +++¹ C190A; V196T; D199F; E203G;   M206I; 75/76 H40R; I93A; A94T; V153I; C190A; +++¹ L195M; V196T; D199F; E203G;  M206I; S207C;  77/78 H40R; I93A; A94S; Q96S; V153L; +++¹ C190A; V196T;D199F; A202G;   E203G; M206I;  79/80 H40R; I93A; A94S; Q96P; V153L; +++¹C190A; V196T; D199F; A202G;   E203G; M206I;  81/82 H40R; I93A; A94S;Q96A; V153L; ++++¹ C190A; V196T; D199F; A202G;   E203G; M206I;  83/84H40R; I93A; A94S; Q96T; V153L; +++¹ C190A; V196T; D199F; A202G;   E203G;M206I;  85/86 H40R; I93A; A94S; Q96N; V153L; +++¹ C190A; V196T; D199F;A202G;   E203G; M206I; R211K;  87/88 H40R; I93A; A94S; Q96N; V153L; +++¹A155C; C190A; V196T; D199F;   A202G; E203G; M206I;  89/90 H40R; I93A;A94S; Q96N; V153L; +++¹ C190A; V196T; D199F; G201I;   A202G; E203G;M206I;  91/92 H40R; I93A; A94S; Q96N; V153L; +++¹ C190A; V196T; D199F;G201L;   A202G; E203G; M206I;  93/94 H40R; I93A; A94S; Q96N; V153L; +++¹C190A; V196T; D199F; G201A;   A202G; E203G; M206I;  95/96 H40R; I93A;A94S; L95V; Q96N; +++¹ R127Q; V153L; C190A; V196T;   D199F; A202G;E203G; M206I; R211K;  97/98 H40R; A94S; L95M; Q96N; L147M; +++¹ V153L;C190A; V196T; D199F;   G201I; A202G; E203G; M206I;  99/100 H40R; A94S;Q96P; V153L; C190A; ++++² V196T; D199F; G201A; A202G; E203G; M206I;R211K; 101/102 L21F; D25R; H40R; I93A; A94S; ++++² Q96P; R108K; S117G;L147M; V153L; C190A; V196T; D199F; A202G; E203G; M206I; N221D; 103/104L21R; D25R; H40R; I93A; A94S; ++++² Q96P; R108K; S117G; L147M; V153L;C190A; V196T; D199F; A202G; E203G; M206I; N221D; 105/106 L21F; D25R;H40R; I93A; A94S; +++++² Q96P; R108K; R127K; L147M; V153L; C190A; V196T;D199F; A202G; E203G; M206I; N221D; 107/108 L21R; D25R; H40R; I93A; A94S;++++² Q96P; R108K; S117G; L147M; V153L; C190A; V196T; D199F; A202G;E203G; M206I; 109/110 L21F; D25R; H40R; I93A; A94S; ++++² Q96P; R108K;S117G; L147M; V153L; V163I; C190A; V196T; D199F; A202G; E203G; M206I;N221D; 111/112 L21R; H40R; I93A; A94S; Q96P; ++++² S117G; L147M; V153L;C190A; V196T; D199F; A202G; E203G; M206I; N221D; 113/114 L21R; H40R;I93A; A94S; Q96P; ++++² S117G; R127K; L147M; V153L; C190A; V196T; D199F;A202G; E203G; M206I; N221D; 115/116 H40R; I93A; A94S; Q96P; S117G; ++++²L147M; V153L; C190A; V196T; D199F; A202G; E203G; M206I; N221D; 117/118D25R; H40R; I93A; A94S; Q96P; ++++² R108D; S117G; R127K; L147M; V153L;C190A; V196T; D199F; A202G; E203G; M206I; N221D; 119/120 H40R; I93A;A94S; Q96P; S117G; ++++² L147M; V153L; C190A; V196T; D199F; A202G;E203G; M206I; 121/122 H40R; I93A; A94S; Q96P; R108K; ++++² S117G; R127K;L147M; V153L; C190A; V196T; D199F; A202G; E203G; M206I; N221D; 123/124L21R; D25R; H40R; I93A; A94S; ++++² Q96P; S117G; R127K; V153L; C190A;V196T; D199F; A202G; E203G; M206I; N221D; 125/126 D25R; H40R; I93A;A94S; Q96P; ++++² S117G; R127K; V153L; C190A; V196T; D199F; A202G;E203G; M206I; 127/128 D25R; H40R; I93A; A94S; Q96P; +++++² R108K; S117G;R127K; L147M; V153L; C190A; V196T; D199F; A202G; E203G; M206I; N221D;129/130 D25R; H40R; I93A; A94S; Q96P; ++++² L147M; V153L; C190A; V196T;D199F; A202G; E203G; M206I; N221D; 131/132 L21R; D25R; H40R; I93A; A94S;++++² Q96P; S117G; L147M; V153L; C190A; V196T; D199F; A202G; E203G;M206I; N221D; 133/134 H40R; I93T; A94S; Q96P; V153L; ++++² C190A; V196T;D199F; A202G; E203G; M206I; 135/136 H40R; I93A; Q96P; V153L; C190A;++++² V196T; D199F; A202G; E203G; M206I; 137/138 H40R; I93A; A94S; Q96P;V153L; ++++² C190A; V196T; D199F; A202G; E203G; M205V; M206I; 139/140H40R; A94S; Q96P; S117A; V153L; +++++³ C190A; V196T; D199F; G201A;A202G; E203G; M206I; R211K; 141/142 H40R; A94S; Q96P; S117G; V153L;++++³ C190A; V196T; D199F; G201A; A202G; E203G; M206I; R211K; 143/144H40R; A94S; Q96P; V153L; C190A; +++++³ V196T; D199W; G201A; A202G;E203G; M206I; R211K; 145/146 H40R; A94S; Q96P; L147M; V153L; ++++³C190A; V196T; D199F; G201A; A202G; E203G; M206I; R211K; 147/148 H40R;A64V; A94S; Q96P; V153L; ++++³ C190A; V196T; D199F; G201A; A202G; E203G;M206I; R211K; 149/150 H40R; A94S; Q96P; V153L; C190A; ++++³ V196T;D199F; G201A; A202G; E203G; M206I; R211K; V223I; 151/152 H40R; A94S;Q96P; V153L; C190A; ++++³ L195M; V196T; D199F; G201A; A202G; E203G;M206I; R211K; 153/154 H40R; A94S; Q96P; L147I; V153L; ++++³ C190A;V196T; D199F; G201A; A202G; E203G; M206I; R211K; 155/156 H40R; A94S;Q96P; V99L; R108H; +++++³ L147I; V153L; C190A; V196T; D199F; G201A;A202G; E203G; M205V; M206I; R211K; I226V; 157/158 H40R; A94S; Q96P;V99L; R108H; +++++³ L147I; V153L; C190A; V196T; D199F; G201A; A202G;E203G; M206I; R211K; I226V; 159/160 H40R; A94S; Q96P; V99L; L147I;+++++³ V153L; C190A; V196T; D199F; G201A; A202G; E203G; M205V; M206I;R211K; 161/162 H40R; A94S; Q96P; V99L; R108H; +++++³ S117G; L147I;V153L; C190A; V196T; D199F; G201A; A202G; E203G; M206I; R211K; I226V;163/164 H40R; A94S; Q96P; V99L; R108H; +++++³ L147I; V153L; C190A;V196T; D199F; G201A; A202G; E203G; M205V; M206I; R211K; 165/166 H40R;A94S; Q96P; V99L; L147I; ++++³ V153L; C190A; V196T; D199F; G201A; A202G;E203G; M205V; M206I; R211K; I226V; 167/168 H40R; A64V; A94S; Q96P; V99L;++++++⁴ R108H; S117G; L147I; V153L; C190A; V196T; D199W; G201A; A202G;E203G; M206I; R211K; I226V; ¹Modified final assay conditions: [compound(2)] = 4 g/L, [NADP] = 0.5 g/L, Solvent = toluene:IPA:acetone: buffer(100 mM TEA buffer, pH 7.0 containing 1 mM MgSO₄) in relative % volumesof 30:18:2:50; plate shaken for 2 h at RT. ²Modified final assayconditions: [compound (2)] = 50 g/L, [NADP] = 0.1 g/L, Solvent =toluene:IPA:acetone:buffer (100 mM TEA buffer, pH 7.0 containing 1 mMMgSO₄) in relative % volumes of 30:18:2:50; plate shaken for 24 hat 30°C.. ³Modified final assay conditions: [compound (2)] = 4 g/L, [NADP] =0.1 g/L, Solvent = (IPA + acetone):buffer (100 mM TEA buffer, pH 7.0containing 1 mM MgSO₄) in relative % volumes of 70:30; plate shaken for24 h at RT.  ⁴Modified final assay conditions: [compound (2)] = 80 g/L,[NADP] = 0.1 g/L, Solvent = (IPA + acetone):buffer (100 mM TEA buffer,pH 7.0 containing 1 mM MgSO₄) in relative % volumes of 65:35; plateshaken for 24 h at RT.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure which are capable of converting compound (2) tocompound (1) comprising an amino acid sequence selected from any one ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, or 168. As shown above in Table 2, each of thesepolypeptides comprises one or more amino acid residue differences ascompared to SEQ ID NO: 2, and has at least 2-fold, at least 10-fold, atleast 25-fold, at least 40-fold, or at least 60-fold increased activityrelative to the activity of the reference polypeptide of SEQ ID NO: 2.Specific amino acid differences are shown in Table 2 and include one ormore residue differences as compared to SEQ ID NO:2 at the followingresidue positions: X21, X25, X40, X64, X93, X94, X95, X96, X99, X108,X117, X127, X147, X148, X150, X152, X153, X155, X190, X195, X196, X201,X202, X203, X204, X205, X206, X207, X211, X221, X223, and X226.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, and comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, and furthercomprises a set of amino acid residue differences as compared to SEQ IDNO:2 of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168. In some embodiments, inaddition to the set of amino acid residue differences of any one of thenon-naturally occurring polypeptides of SEQ ID NO: 4 through SEQ ID NO:168, the sequence of the non-naturally occurring polypeptide can furthercomprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residuedifferences at other amino acid residue positions as compared to the SEQID NO: 2. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to SEQ ID NO: 2.

The present disclosure also contemplates a non-naturally occurringpolypeptide capable of converting compound (2) to compound (1) withimproved properties relative to the activity of the polypeptide of SEQID NO: 2, wherein the non-naturally occurring polypeptide comprises anamino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2,and further comprises a set of amino acid residue differences ascompared to SEQ ID NO:2, wherein the amino acid differences are based onlocations or regions in the structure of reference polypeptide (e.g.,SEQ ID NO: 2) and/or the associated function properties. Accordingly,referring to Table 3, a non-naturally occurring or engineeredketoreductase polypeptide of the present disclosure can include an aminoacid substitution at a particular residue at a location in the structureof the reference polypeptide as identified in Table 3. Exemplarysubstitutions at each of the relevant locations include those identifiedin Table 2.

TABLE 3 Structural locations useful for engineered ketoreductasepolypeptides Position Structural location Associated functionalproperties X21 Surface Thermostability X25 Surface Solvent stability X40NADPH-Binding Site Tight binding of NADPH to enzyme X64 NADPH-BindingSite Interacts with NADPH Adenine ring X93 Second sphere active siteThermostability X94 Second sphere active site Thermostability X95 Secondsphere active site Activity X96 Second sphere active site Activity X99Dimer-tetramer interface Thermostability/Solvent stability X108Dimer-tetramer interface Thermostability/Solvent stability X117 CoreThermostability/Solvent stability X127 Second sphere active siteThermostability/Solvent stability X147 Dimer-tetramer interfaceThermostability/Solvent stability X148 Dimer-tetramer interfaceThermostability/Solvent stability X150 Active site Activity X152 Secondsphere active site Activity X153 Second sphere active site Activity X155Position interacting with 95 Activity X190 Active site Activity X195Second sphere active site Thermostability X196 Active site Activity X201Active site Activity X202 Flexible loop Activity X203 Flexible loopActivity X204 Flexible loop Activity X205 Flexible loop Activity X206Flexible loop Activity X207 Flexible loop Activity X211 Second sphereactive site Activity X221 Surface Thermostability/Solvent stability X223Core Thermostability/Solvent stability X226 Dimer-tetramer interfaceThermostability/Solvent stability

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, which comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 and atleast the following features: residue at position corresponding to X40is R; residue at position corresponding to X153 is I, or L; residue atposition corresponding to X190 is A or P; residue at positioncorresponding to X196 is T; residue at position corresponding to X199 isF, or W; and residue at position corresponding to X206 is I. In someembodiments, the amino acid sequence further comprises at least onefeature or group of features selected from: (a) residue at position X93is A and residue at position X94 is T; (b) residue at position X93 is Aand residue at position X94 is S; (c) residue at position X93 is A andresidue at position X94 is S; (d) residue at position X93 is I andresidue at position X94 is S; (e) residue at position X203 is G; (f)residue at position X202 is G and residue at position X203 is G; or (f)residue at position X201 is A, residue at position X202 is G, andresidue at position X203 is G.

In some embodiments, any of the non-naturally occurring polypeptides ofthe present disclosure capable of converting compound (2) to compound(1) with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2 and an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 2, can further one or morefeatures selected from: residue at position corresponding to X21 is R orF; residue at position corresponding to X25 is R, T, or N; residue atposition corresponding to X40 is R; residue at position corresponding toX64 is V; residue at position corresponding to X93 is A; residue atposition corresponding to X94 is T, S, or P; residue at positioncorresponding to X95 is V, or M; residue at position corresponding toX96 is V, G, A, N, S, P, or T; residue at position corresponding to X99is L; residue at position corresponding to X108 is H; residue atposition corresponding to X117 is A, or G; residue at positioncorresponding to X127 is K, or Q; residue at position corresponding toX147 is M, or I; residue at position corresponding to X148 is I; residueat position corresponding to X150 is H, or A; residue at positioncorresponding to X152 is N, or F; residue at position corresponding toX153 is I, or L; residue at position corresponding to X155 is C; residueat position corresponding to X190 is A; residue at positioncorresponding to X195 is M; residue at position corresponding to X196 isT, A, S, C, or N; residue at position corresponding to X199 is F, or W;residue at position corresponding to X201 is I, L, or A; residue atposition corresponding to X202 is L, N, V, or G; residue at positioncorresponding to X203 is G; residue at position corresponding to X204 isV, or A; residue at position corresponding to X205 is V; residue atposition corresponding to X206 is I; residue at position correspondingto X207 is T, C, I, or N; residue at position corresponding to X211 isK; residue at position corresponding to X221 is D; residue at positioncorresponding to X223 is I; residue at position corresponding to X226 isV.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, which comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, and atleast the following features: residue at position corresponding to X40is R; residue at position corresponding to X153 is I, or L; residue atposition corresponding to X190 is A or P; residue at positioncorresponding to X196 is T; residue at position corresponding to X199 isF, or W; and residue at position corresponding to X206 is I, wherein theamino acid sequence further comprises at least one of the following setsof features (a) through (h):

(a) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is A; residue at position corresponding to X94 isT; residue at position corresponding to X153 is I; residue at positioncorresponding to X190 is A; residue at position corresponding to X196 isT; residue at position corresponding to X199 is F; residue at positioncorresponding to X203 is G; and residue at position corresponding toX206 is I; or

(b) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is A; residue at position corresponding to X94 isS; residue at position corresponding to X96 is N; residue at positioncorresponding to X153 is L; residue at position corresponding to X190 isA; residue at position corresponding to X196 is T; residue at positioncorresponding to X199 is F; residue at position corresponding to X202 isG; residue at position corresponding to X203 is G; and residue atposition corresponding to X206 is I; or

(c) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is A; residue at position corresponding to X94 isS; residue at position corresponding to X96 is A, P, or N; residue atposition corresponding to X153 is L; residue at position correspondingto X190 is A; residue at position corresponding to X196 is T; residue atposition corresponding to X199 is F; residue at position correspondingto X202 is G; residue at position corresponding to X203 is G; andresidue at position corresponding to X206 is I; and, optionally furthercomprises: residue at position corresponding to X96 is N; and residue atposition corresponding to X201 is I, or L; or

(d) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is A; residue at position corresponding to X96 isP; residue at position corresponding to X153 is L; residue at positioncorresponding to X190 is A; residue at position corresponding to X196 isT; residue at position corresponding to X199 is F; residue at positioncorresponding to X202 is G; residue at position corresponding to X203 isG; and residue at position corresponding to X206 is I; and optionallyfurther comprises: residue at position corresponding to X21 is F;residue at position corresponding to X25 is R; residue at positioncorresponding to X94 is S; residue at position corresponding to X96 isP; residue at position corresponding to X108 is K; residue at positioncorresponding to X127 is K; residue at position corresponding to X147 isM; residue at position corresponding to X153 is L; residue at positioncorresponding to X205 is V; residue at position corresponding to X211 isK; and residue at position corresponding to X221 is D; or

(e) residue at position corresponding to X21 is F; residue at positioncorresponding to X25 is R; residue at position corresponding to X40 isR; residue at position corresponding to X93 is A; residue at positioncorresponding to X94 is S; residue at position corresponding to X96 isP; residue at position corresponding to X108 is K; residue at positioncorresponding to X127 is K; residue at position corresponding to X147 isM; residue at position corresponding to X153 is L; residue at positioncorresponding to X190 is A; residue at position corresponding to X196 isT; residue at position corresponding to X199 is F; residue at positioncorresponding to X202 is G; residue at position corresponding to X203 isG; residue at position corresponding to X206 is I; and residue atposition corresponding to X221 is D; or

(f) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is I; residue at position corresponding to X94 isS; residue at position corresponding to X96 is P; residue at positioncorresponding to X153 is L; residue at position corresponding to X190 isA; residue at position corresponding to X196 is T; residue at positioncorresponding to X199 is F, or W; residue at position corresponding toX201 is A; residue at position corresponding to X202 is G; residue atposition corresponding to X203 is G; residue at position correspondingto X206 is I; and residue at position corresponding to X211 is K; andoptionally further comprises: residue at position corresponding to X64is V; residue at position corresponding to X99 is L; residue at positioncorresponding to X108 is H; residue at position corresponding to X117 isA, or G; residue at position corresponding to X147 is I, or M; residueat position corresponding to X195 is M; residue at positioncorresponding to X205 is V; residue at position corresponding to X223 isI; and residue at position corresponding to X226 is V; or

(g) residue at position corresponding to X40 is R; residue at positioncorresponding to X93 is I; residue at position corresponding to X94 isS; residue at position corresponding to X96 is P; residue at positioncorresponding to X99 is L; residue at position corresponding to X108 isH; residue at position corresponding to X117 is G; residue at positioncorresponding to X147 is I; residue at position corresponding to X153 isL; residue at position corresponding to X190 is A; residue at positioncorresponding to X196 is T; residue at position corresponding to X199 isF; residue at position corresponding to X201 is A; residue at positioncorresponding to X202 is G; residue at position corresponding to X203 isG; residue at position corresponding to X206 is I; residue at positioncorresponding to X211 is K; and residue at position corresponding toX226 is V; or

(h) residue at position corresponding to X40 is R; residue at positioncorresponding to X64 is V; residue at position corresponding to X93 isI; residue at position corresponding to X94 is S; residue at positioncorresponding to X96 is P; residue at position corresponding to X99 isL; residue at position corresponding to X108 is H; residue at positioncorresponding to X117 is G; residue at position corresponding to X147 isI; residue at position corresponding to X153 is L; residue at positioncorresponding to X190 is A; residue at position corresponding to X196 isT; residue at position corresponding to X199 is W; residue at positioncorresponding to X201 is A; residue at position corresponding to X202 isG; residue at position corresponding to X203 is G; residue at positioncorresponding to X206 is I; residue at position corresponding to X211 isK; and residue at position corresponding to X226 is V.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise a sequence having one or more amino acidresidue differences as compared to SEQ ID NO: 2 at residue positionsaffecting activity for conversion of compound (2) to compound (1), whichpositions include the following: X21; X25; X64; X93; X94; X95; X96; X99;X108; X117; X127; X147; X148; X150; X152; X153; X155; X163; X190; X195;X196; X199; X201; X202; X203; X204; X205; X206; X207; X211; X221; X223;and X226. In some embodiments, the specific amino acid differencesresulting in increased activity for conversion of compound (2) tocompound (1) relative to the reference polypeptide of SEQ ID NO: 2 canbe selected from the following: residue at position corresponding to X21is F or R; residue at position corresponding to X25 is N, R, or T;residue at position corresponding to X64 is V; residue at positioncorresponding to X93 is A or T; residue at position corresponding to X94is P, S, or T; residue at position corresponding to X95 is M or V;residue at position corresponding to X96 is A, N, G, P, S, T, or V;residue at position corresponding to X99 is L; residue at positioncorresponding to X108 is D, H, or K; residue at position correspondingto X117 is A or G; residue at position corresponding to X127 is K or Q;residue at position corresponding to X147 is I or M; residue at positioncorresponding to X148 is I; residue at position corresponding to X150 isH; residue at position corresponding to X152 is N or F; residue atposition corresponding to X153 is I or L; residue at positioncorresponding to X155 is C; residue at position corresponding to X163 isI; residue at position corresponding to X190 is A; residue at positioncorresponding to X195 is M; residue at position corresponding to X196 isA, C, N, S, or T; residue at position corresponding to X199 is F or W;residue at position corresponding to X201 is A, I, or L; residue atposition corresponding to X202 is G, L, N, or V; residue at positioncorresponding to X203 is G; residue at position corresponding to X204 isA or V; residue at position corresponding to X205 is V; residue atposition corresponding to X206 is I; residue at position correspondingto X207 is C, I, N, or T; residue at position corresponding to X211 isK; residue at position corresponding to X221 is D; residue at positioncorresponding to X223 is I; and residue at position corresponding toX226 is V.

In some embodiments, the non-naturally occurring polypeptides capable ofconverting compound (2) to compound (1) can have increasedthermostability as compared to the polypeptide of SEQ ID NO: 2 (oranother reference polypeptide, e.g., SEQ ID NO: 80 or 100).Thermostability can be determined by preincubating the polypeptide at adefined temperature and time, e.g., 4° C.-46° C. for 18-24 hours,followed by measuring the % residual activity using a defined assay.Exemplary preincubation conditions include preincubation at 30° C. for18 h, or 40° C. for 24 h. Accordingly, in some embodiments, thenon-naturally occurring polypeptides of the present disclosure cancomprise an amino acid sequence having one or more residue differencesas compared to SEQ ID NO: 2 at residue positions affectingthermostability, which positions include the following: X21; X93; X94;X117; X127; X147; X195; and X199. In some embodiments, specific aminoacid differences resulting in increased thermostability relative to thereference polypeptide of SEQ ID NO: 2 can be selected from the followingsubstitutions: residue at position corresponding to X21 is F; residue atposition corresponding to X93 is T; residue at position corresponding toX94 is A; residue at position corresponding to X117 is G or A; residueat position corresponding to X127 is K; residue at positioncorresponding to X147 is I; residue at position corresponding to X195 isM; and residue at position corresponding to X199 is W.

In some embodiments, the present disclosure provides non-naturallyoccurring polypeptides capable of converting compound (2) to compound(1) and having at least 1.5-fold, 2.5-fold, 5-fold, 7.5-fold or moreincreased thermostability following 18 h preincubation at 40° C. ascompared to the polypeptide of SEQ ID NO: 80 or SEQ ID NO: 100, whichcomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 80 or SEQ ID NO: 100, and at least one of the followingsubstitutions: residue at position corresponding to X21 is F; residue atposition corresponding to X93 is T; residue at position corresponding toX94 is A; residue at position corresponding to X117 is G or A; residueat position corresponding to X127 is K; residue at positioncorresponding to X147 is I; residue at position corresponding to X195 isM; and residue at position corresponding to X199 is W.

In some embodiments, the present disclosure provides non-naturallyoccurring polypeptides capable of converting compound (2) to compound(1) and having at least 1.5-fold, 2.5-fold, 5-fold, 7.5-fold or moreincreased thermostability following 18 h preincubation at 40° C. ascompared to the polypeptide of SEQ ID NO: 80 or SEQ ID NO: 100, whichcomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 80 or SEQ ID NO: 100, and the set of amino acid residuedifferences of any one of the non-naturally occurring polypeptides ofSEQ ID NOs: 134, 136, 140, 142, 144, 152, 154, 156, 158, 160, 162, 164,or 166.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise an amino acid sequence having residuedifferences as compared to SEQ ID NO: 2 at residue positions affectingsolvent stability, which positions include the following: X25; X147; andX221. In some embodiments, specific amino acid differences resulting inincreased solvent stability relative to the reference polypeptide of SEQID NO: 2 (e.g., increased activity relative to SEQ ID NO: 2 in up to 65%isopropanol) can be selected from the following substitutions: residueat position corresponding to X25 is R; residue at position correspondingto X147 is M; and residue at position corresponding to X221 is D.

In some embodiments, the non-naturally occurring polypeptides of thepresent disclosure can comprise an amino acid sequence having residuedifferences as compared to SEQ ID NO: 2 at residue positions affectingcofactor binding, which positions include X40. In some embodiments,specific amino acid differences affecting cofactor binding can beselected from the following: residue at position corresponding to X40 isR.

As will be apparent to the skilled artisan, various combinations ofresidue differences as compared to SEQ ID NO:2 at residue positionsaffecting enzymatic activity, thermostability, solvent stability, andcofactor binding can be made to form the polypeptides of the presentdisclosure.

In addition to the residue positions specified above, any of thenon-naturally occurring ketoreductase polypeptides disclosed herein canfurther comprise other residue differences relative to SEQ ID NO:2 atother residue positions. Residue differences at these other residuepositions provide for additional variations in the amino acid sequencewithout adversely affecting the ability of the polypeptide to carry outthe conversion of compound (2) to compound (1). hi some embodiments, thepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40 residue differences at other amino acid residuepositions as compared to the reference sequence. In some embodiments,the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residue differences atother residue positions. The residue difference at these other positionscan include conservative changes or non-conservative changes. In someembodiments, the residue differences can comprise conservativesubstitutions and non-conservative substitutions as compared to thewild-type ketoreductase of SEQ ID NO: 2.

Amino acid residue differences at other positions relative to SEQ ID NO:2 or the wild-type L. kefir ketoreductase sequence (Genbank acc. No.AAP94029.1; GI: 33112056) and the affect of these differences on enzymefunction are provide by e.g., engineered ketoreductase polypeptides inthe following patent publications, each of which is hereby incorporatedby reference herein: US 20080318295A1; US 20090093031A1; US20090155863A1; US 20090162909A1; US 20090191605A1; US 20100055751A1;WO/2010/025238A2; WO/2010/025287A2; and US 20100062499A1. Accordingly,in some embodiments, one or more of the amino acid differences providedin the engineered ketoreductase polypeptides of these publications couldalso be introduced into a non-naturally occurring polypeptide of thepresent disclosure.

In some embodiments, the present disclosure provides a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, which comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, with theproviso that the amino acid sequence of any one or more of theketoreductase polypeptides disclosed in any one or more of the followingpatent publications are excluded: US 20080318295A1; US 20090093031A1; US20090155863A1; US 20090162909A1; US 20090191605A1; US 20100055751A1;WO/2010/025238A2; WO/2010/025287A2; US 20100062499A1; and WO2008/151324A1.

In some embodiments, the polypeptides can comprise deletions of theengineered ketoreductase polypeptides described herein. Thus, for eachand every embodiment of the polypeptides of the disclosure, thedeletions can comprise one or more amino acids, 2 or more amino acids, 3or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 8 or more amino acids, 10 or more amino acids, 15 ormore amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, up to 10% of the total number of amino acids, upto 20% of the total number of amino acids of the polypeptides, as longas the functional activity of the polypeptide with respect to theconversion of compound (2) to compound (1) is present. In someembodiments, the deletions can comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-26, 1-30, 1-35, or 1-40 amino acid residues. In some embodiments, thenumber of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35, or 40 amino acids. In someembodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 amino acid residues.

In some embodiments, the polypeptides can comprise fragments of theengineered polypeptides described herein. In some embodiments, thefragments can have about 80%, 90%, 95%, 98%, and 99% of the full-lengthpolypeptide, e.g., the polypeptide of SEQ ID NO:2, as long as thefunctional activity of the polypeptide with respect to the conversion ofcompound (2) to compound (1) is present.

In some embodiments, the polypeptides of the disclosure can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purifications sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

As will be understood by the skilled artisan, the polypeptides describedherein are not restricted to the genetically encoded amino acids. Inaddition to the genetically encoded amino acids, the polypeptidesdescribed herein may be comprised, either in whole or in part, ofnaturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the polypeptidesdescribed herein may be comprised include, but are not limited to: theD-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the polypeptide described herein can be provided inthe form of kits. The enzymes in the kits may be present individually oras a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the polypeptides can be provided on a substrate. Insome embodiments, the polypeptides can be provided in the form of anarray in which the polypeptides are arranged in positionally distinctlocations. The array can be used to test a variety of aryl alkylsulfides for conversion by the polypeptides. “Substrate,” “support,”“solid support,” “solid carrier,” or “resin” in the context of arraysrefer to any solid phase material. Substrate also encompasses terms suchas “solid phase,” “surface,” and/or “membrane.” A solid support can becomposed of organic polymers such as polystyrene, polyethylene,polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide,as well as co-polymers and grafts thereof. A solid support can also beinorganic, such as glass, silica, controlled pore glass (CPG), reversephase silica or metal, such as gold or platinum. The configuration of asubstrate can be in the form of beads, spheres, particles, granules, agel, a membrane or a surface. Surfaces can be planar, substantiallyplanar, or non-planar. Solid supports can be porous or non-porous, andcan have swelling or non-swelling characteristics. A solid support canbe configured in the form of a well, depression, or other container,vessel, feature, or location. A plurality of supports can be configuredon an array at various locations, addressable for robotic delivery ofreagents, or by detection methods and/or instruments.

In certain embodiments, the kits of the present disclosure includearrays comprising a plurality of different engineered ketoreductasepolypeptides at different addressable position, wherein the differentpolypeptides are different variants of a reference sequence each havingat least one different improved enzyme property. Such arrays comprisinga plurality of engineered polypeptides and methods of their use aredescribed in, e.g., WO2009/008908A2.

5.4 Ketoreductase Polynucleotides, Expression Vectors, and Host Cells

In another aspect, the present disclosure provides polynucleotidesencoding the non-naturally occurring or engineered polypeptidesdescribed herein. These polynucleotides may be operatively linked to oneor more heterologous regulatory sequences that control gene expressionto create a recombinant polynucleotide capable of expressing theketoreductase polypeptide. Expression constructs containing aheterologous polynucleotide encoding the engineered ketoreductasepolypeptide can be introduced into appropriate host cells to express thecorresponding polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thus, havingidentified a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in Table 2.

In some embodiments, the polynucleotides can be selected and/orengineered to comprise codons that are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. Since not all codons need to be replaced to optimize the codonusage of the ketoreductases (e.g., because the natural sequence can havepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues), codon optimized polynucleotides encodingthe ketoreductase polypeptides may contain preferred codons at about40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of thefull length coding region.

In some embodiments, the polynucleotide encodes a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, and comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the polynucleotide encodes a non-naturallyoccurring polypeptide capable of converting compound (2) to compound (1)with at least 2-fold, at least 10-fold, at least 25-fold, at least40-fold, or at least 60-fold increased activity relative to the activityof the polypeptide of SEQ ID NO: 2, and comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, or 168, with the proviso that theamino acid sequence comprises any one of the set of residue differencesas compared to SEQ ID NO: 2 contained in any one of the polypeptidesequences of SEQ ID NO:4 to SEQ ID NO:168 listed in Table 2.

In some embodiments, the polynucleotides encoding the polypeptides areselected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, and 167.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, or 167, or a complement thereof, where the highly stringentlyhybridizing polynucleotides encode a non-naturally occurring polypeptidecapable of converting compound (2) to compound (1) with at least 2-fold,at least 10-fold, at least 25-fold, at least 40-fold, or at least60-fold increased activity relative to the activity of the polypeptideof SEQ ID NO: 2.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered ketoreductasepolypeptides described herein. In some embodiments, the referencepolynucleotide is selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, and 167.

An isolated polynucleotide encoding a non-naturally occurringpolypeptide of the disclosure may be manipulated in a variety of ways toprovide for expression of the polypeptide. In some embodiments, thepolynucleotides encoding the polypeptides can be provided as expressionvectors where one or more control sequences is present to regulate theexpression of the polynucleotides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used.Exemplary bacterial promoters include E. coli lac operon, E. coli trpoperon, bacteriophage□ 1, Streptomyces coelicolor agarase gene (dagA),Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformisalpha-amylase gene (amyL), beta-lactamase gene, and tac promoter;exemplary promoters for filamentous fungal host cells, include promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease, and mutant, truncated, and hybridpromoters thereof, and exemplary yeast cell promoters can be from thegenes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomycescerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase.

In some embodiments, the control sequence may also be a signal peptidecoding region that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The signal sequence typically depends on thetype of host cells being used to express the polypeptide. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NC1B 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Exemplary signal peptide coding regions forfilamentous fungal host cells can be the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase. Useful signal peptides for yeast host cells can befrom the genes for Saccharomyces cerevisiae alpha-factor andSaccharomyces cerevisiae invertase.

Other control sequences, such as leader sequence, polyadenylationsequence, and transcription terminator sequences can use those availablein the art (see Sambrook, supra, and Current Protocols in MolecularBiology, supra).

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered ketoreductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The recombinant expression vector may be anyvector (e.g., a plasmid or virus), which can be conveniently subjectedto recombinant DNA procedures and can bring about the expression of thepolynucleotide sequence. The choice of the vector will typically dependon the compatibility of the vector with the host cell into which thevector is to be introduced. The vectors may be linear or closed circularplasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. The expressionvector preferably contains one or more selectable markers, which permiteasy selection of transformed cells. A selectable marker is a gene theproduct of which provides for biocide or viral resistance, resistance toheavy metals, prototrophy to auxotrophs, resistance to chemical agents(e.g., antibiotics) and the like.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an engineered ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase polypeptide in the host cell. Host cells for use inexpressing the ketoreductase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanomacells; and plant cells. Exemplary host cells are Escherichia coli BL21and W3110.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the ketoreductase may be introduced into host cells byvarious methods known in the art (e.g., electroporation, biolisticparticle bombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion).

In the embodiments herein, the non-naturally occurring or engineeredketoreductase polypeptides and nucleotides encoding such polypeptidescan be prepared using methods commonly used by those skilled in the art.As noted above, the naturally-occurring amino acid sequence andcorresponding polynucleotide encoding the ketoreductase enzyme ofLactobacillus kefir. In some embodiments, the parent polynucleotidesequence is codon optimized to enhance expression of the ketoreductasein a specified host cell.

The engineered ketoreductase polypeptides can be obtained by subjectingthe polynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods (see e.g., Stemmer, 1994,Proc Natl Acad Sci USA 91:10747-10751; PCT Publ. Nos. WO 95/22625, WO97/0078, WO 97/35966, WO 98/27230, WO 00/42651, and WO 01/75767; U.S.Pat. Nos. 6,537,746, 6,117,679, 6,376,246, and 6,586,182; and U.S. Pat.Publ. Nos. 20080220990A1 and 20090312196A1; each of which is herebyincorporated by reference herein).

Other directed evolution procedures that can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Zhaoet al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell etal., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis(Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesisand directed evolution techniques useful for the purposes herein arealso described in the following references: Ling, et al., 1997, Anal.Biochem. 254(2):157-78; Dale et al., 1996, Methods Mol. Biol. 57:369-74;Smith, 1985, Ann. Rev. Genet. 19:423-462; Botstein et al., 1985, Science229:1193-1201; Carter, 1986, “Site-directed mutagenesis,” Biochem. J.237:1-7; Kramer et al., 1984, Cell 38:879-887; Wells et al., 1985, Gene34:315-323; Minshull et al., 1999, Curr Opin Chem Biol 3:284-290;Christians et al., 1999, Nature Biotech 17:259-264; Crameri et al.,1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; and Stemmer, 1994,Nature 370:389-391. All publications are incorporated herein byreference.

In some embodiments, the clones obtained following mutagenesis treatmentare screened for non-naturally occurring ketoreductases having a desiredenzyme property. Measuring ketoreductase enzyme activity from theexpression libraries can be performed using the standard techniques,such as separation of the product (e.g., by HPLC) and detection of theproduct by measuring UV absorbance of the separated substrate andproducts and/or by detection using tandem mass spectroscopy (e.g.,MS/MS). Clones containing a polynucleotide encoding the desiredengineered polypeptides are then isolated, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Exemplary assays are provided below in Example 3.

Where the sequence of the polypeptide is known, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods, e.g., the classicalphosphoramidite method described by Beaucage et al., 1981, Tet Lett22:1859-69, or the method described by Matthes et al., 1984, EMBO J.3:801-05. In some embodiments, fragments of up to about 100 bases can beindividually synthesized, then joined (e.g., by enzymatic or chemicallitigation methods, or polymerase mediated methods) to form any desiredcontinuous sequence.

In some embodiments, the present disclosure also provides methods forpreparing or manufacturing the non-naturally occurring polypeptidescapable of converting compound (2) to compound (1), wherein the methodscomprise: (a) culturing a host cell capable of expressing apolynucleotide encoding the non-naturally occurring polypeptide and (b)isolating the polypeptide from the host cell. The non-naturallyoccurring polypeptides can be expressed in appropriate cells (asdescribed above), and isolated (or recovered) from the host cells and/orthe culture medium using any one or more of the well known techniquesused for protein purification, including, among others, lysozymetreatment, sonication, filtration, salting-out, ultra-centrifugation,and chromatography. Chromatographic techniques for isolation of theketoreductase polypeptide include, among others, reverse phasechromatography high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, and affinity chromatography.

In some embodiments, the non-naturally occurring polypeptide of thedisclosure can be prepared and used in various isolated forms includingbut not limited to crude extracts (e.g., cell-free lysates), powders(e.g., shake-flask powders), lyophilizates, and substantially purepreparations (e.g., DSP powders), as further illustrated in the Examplesbelow.

In some embodiments, the non-naturally occurring polypeptide of thedisclosure can be prepared and used in purified form. Generally,conditions for purifying a particular enzyme will depend, in part, onfactors such as net charge, hydrophobicity, hydrophilicity, molecularweight, molecular shape, etc., and will be apparent to those havingskill in the art. To facilitate purification, it is contemplated that insome embodiments the engineered ketoreductase polypeptides of thepresent disclosure can be expressed as fusion proteins with purificationtags, such as His-tags having affinity for metals, or antibody tags forbinding to antibodies, e.g., myc epitope tag.

5.5 Methods of Use

The engineered ketoreductase polypeptides described herein can be usedin processes comprising the conversion of compound (2) to compound (1)as shown in Scheme 1, for example in process for manufacturing compound(1), which is used as the active pharmaceutical ingredient, Ezetimibe.Furthermore, the biocatalytic abilities of the non-naturally occurringketoreductase polypeptides disclosure are not limited to the conversionof compound (2) to compound (1). Additionally, the engineeredketoreductase polypeptides described herein can be used for theconversion of analogs of compound (2) to the corresponding chiralalcohol analogs of compound (1) in diastereomeric excess.

In some embodiments, the disclosure provides methods for preparingcompound (1) or an analog of compound (1) in diastereomeric excesscomprising: contacting compound (2) or an analog of compound (2) with anengineered polypeptide of the present disclosure (e.g., as described inTable 2 and elsewhere herein) in the presence of NADPH or NADH cofactorunder suitable reaction conditions. Suitable reactions conditions forthe conversion of compound (2) to compound (1), or the conversion of ananalog of compound (2) to the corresponding analog of compound (1),using the engineered polypeptides of the present disclosure aredescribed in greater detail below and some exemplary suitable reactionconditions also are provided in the Examples.

The engineered polypeptides of the present disclosure of improvedenzymatic properties for the conversion of compound (2) to compound (1)relative to the naturally occurring ketoreductase polypeptide of SEQ IDNO: 2, including increased conversion rates, increased stereoselectivity(resulting in compound (1) in greater diastereomeric excesses),increased solvent stability, and increased thermal stability.Accordingly, it is contemplated that any of the engineered polypeptidesdisclosed herein may be used in improved methods that comprise theconversion of compound (2) to compound (1). For example, in someembodiments, the methods of the present disclosure can be carried outwherein the engineered polypeptide is selected from an amino acidsequence having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to SEQ ID NO: 2, which further comprises the combinationof residue differences compared to SEQ ID NO: 2 of any one of engineeredpolypeptides disclosed in Table 2 (e.g., even-numbered SEQ ID NOs:4-168). In some embodiments, the any one or more of the polypeptides ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, or 168 may be used in the methods disclosed herein.

The present disclosure also contemplates ranges of suitable reactionconditions that can be used in the methods, including but not limited toranges of pH, temperature, buffer, solvent system, substrate loading,polypeptide loading, cofactor loading, atmosphere, and reaction time.The present disclosure also contemplates that the methods comprising thebiocatalytic conversion compound (2) to compound (1) using an engineeredpolypeptide of the disclosure can further comprise chemical steps ofcompound (1) product work-up, extraction, isolation, purification,and/or crystallization, each of which can be carried out under a rangeof conditions.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise compound (2) substrate loading of at least about 20 g/L, about40 g/L, about 50 g/L, about 75 g/L, about 100 g/L, about 200 g/L, about250 g/L, about 300 g/L, about 400 g/L, or even greater. In certainembodiments, methods for preparing compound (1) of the presentdisclosure can be carried out wherein the reaction conditions comprisecompound (2) substrate loading of about 50-100 g/L, about 50-200 g/L,about 50-300 g/L, about 50-400 g/L, about 100 g/L, about 200 g/L, about300 g/L or about 400 g/L. The values for substrate loadings providedherein are based on the molecular weight of compound (2), however italso contemplated that the equivalent molar amounts of various hydratesand salts of compound (2) also can be used in the methods.

The improved enzymatic activity of the engineered polypeptides of thepresent disclosure in the conversion of compound (2) to compound (1)provides for methods wherein higher percentage conversion can beachieved with lower concentrations of the engineered polypeptide. Theuse of lower concentration of the engineered polypeptide in a methodcomprising a conversion of compound (2) to compound (1) also reduces theamount of residual protein that may need to be removed in subsequentsteps for purification of compound (1). In some embodiments, the methodsfor preparing compound (1) of the present disclosure can be carried outwherein the reaction conditions comprise an engineered polypeptideconcentration of about 0.1-3.0 g/L, about 0.5-2.75 g/L, about 1.0-2.5g/L, about 1.5-2.5 g/L, about 3 g/L, about 2 g/L, about 1.5 g/L, about1.0 g/L, about 0.75 g/L, or even lower concentration.

In certain embodiments, the temperature of the suitable reactionconditions can be chosen to maximize the reaction rate at highertemperatures while maintaining the activity of the enzyme for sufficientduration for efficient conversion of the substrate to the product. Wherehigher temperatures are used, polypeptides with increasedthermostability can be selected to carry out the process.

The engineered polypeptides of the present disclosure have increasedthermal stability relative to the naturally occurring ketoreductasepolypeptide of SEQ ID NO: 2. This allows the engineered polypeptides tobe used in methods for converting compound (2) to compound (1) at highertemperatures which can result in increased conversion rates and improvedsubstrate solubility characteristics for the reaction, althoughsubstrate or product degradation at higher temperatures can contributeto decreased process yields. In certain embodiments, the method can becarried out wherein the reaction conditions comprise a temperature ofabout 20° C. to about 40° C., about 23° C. to about 37° C., about 25° C.to about 35° C., about 26° C. to about 32° C., or about 28° C. to about30° C. In certain embodiments, the temperature during the enzymaticreaction can be maintained at ambient (e.g., 25° C.), 27° C., 30° C.,32° C., 35° C., 37° C., 40° C.; or in some embodiments adjusted over atemperature profile during the course of the reaction.

In certain embodiments, the methods for preparing compound (1) of thepresent disclosure the pH of the reaction mixture may be maintained at adesired pH or within a desired pH range by the addition of an acid or abase during the course of the reaction. In certain embodiments, the pHof the reaction mixture may change or be changed during the course ofthe reaction. Thus, it is contemplated that in some embodiments the pHmay be controlled by using an aqueous solvent that comprises a buffer.Suitable buffers to maintain desired pH ranges are known in the art andinclude, for example, phosphate buffer, triethanolamine buffer, and thelike. Combinations of buffering and acid or base addition may also beused.

In certain embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise a pH of about 6.0 to about 7.5, a pH of about 6.25 to about7.25, a pH of about 6.5 to about 7.25, a pH of about 6.6 to about 7.25,a pH of about 6.6 to about 7.0, a pH of about 6.75 to about 7.25, or apH of about 6.75. Below pH 6.5 the rate of the biocatalytic conversionof compound (2) to compound (1) slows down and consequently a longeroverall reaction time (e.g., >24 h) may be needed to achieve a highlevel of conversion (e.g., >97%). Also, NADP+ cofactor is less stablebelow pH 6.25. Above pH 7.25 the degradation of the compound (2) andcompound (1) increases, which may result in decreased overall yield andpurity of compound (1) made according to the method.

The methods for preparing compound (1) of the present disclosure aregenerally carried out in a solvent. Suitable solvents include water,aqueous buffer solutions, organic solvents, and/or co-solvent systems,which generally comprise aqueous solvents and organic solvents.

In certain embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise a solution comprising an aqueous buffer solution, an organicsolvent, or a co-solvent system. In some embodiments, the buffersolution is selected from TEA (e.g., about 0.025 M to about 0.25 M TEA)and potassium phosphate (e.g., about 0.025 M to about 0.25 M phosphate).In certain embodiments, the co-solvent system comprises about 30% (v/v)to about 70% (v/v) of an aqueous buffer solution (e.g., about 0.1 M TEA)and about 70% to about 30% of an organic solvent solution (e.g., IPAand/or toluene). In some embodiments, the reaction conditions comprisewater as a suitable solvent with no buffer present.

The engineered polypeptides of the present disclosure have increasedstability to organic solvent relative to the naturally occurringketoreductase polypeptide of SEQ ID NO: 2. This allows the engineeredpolypeptides to be used in methods for converting compound (2) tocompound (1) in co-solvent systems with higher concentrations of organicsolvent which can result in improved product solubility characteristicsand increased percent conversion (e.g., 97% or greater conversion ofcompound (2), at 100 g/L concentration, to compound (1) in 24 h).

In another embodiment, the co-solvent system comprises an aqueous buffersolution and IPA, wherein the IPA concentration is about 25-75% (v/v),about 35-75% (v/v), about 45-75% (v/v), about 55-75% (v/v), about 60-70%(v/v), about 62-68% (v/v), at least about 25% (v/v), at least about 35%(v/v), at least about 45% (v/v), at least about 55% (v/v), at leastabout 65% (v/v), about 60% (v/v), about 65% (v/v), or about 70% (v/v).In certain embodiments, the reaction conditions comprise a co-solventsystem of 0.1 M TEA buffer and about 60% (v/v) to about 70% (v/v) IPA.In certain embodiments, the reaction conditions comprise a co-solventsystem of about 35% (v/v) 0.1 M TEA buffer and about 65% (v/v) IPA.

In some embodiments, the co-solvent system comprises an aqueous buffersolution, IPA, and another organic solvent, such as toluene. In someembodiments, the co-solvent system comprises about 45-55% (v/v) of anaqueous buffer solution, about 25-35% (v/v) IPA, and about 25-35% (v/v)toluene. In certain embodiments, the co-solvent system comprises about50% (v/v) of an aqueous buffer solution (e.g., 0.1 M TEA), about 20%(v/v) IPA, and about 30% (v/v) toluene.

In certain embodiments, the methods comprising the conversion ofcompound (2) to compound (1) can be carried out wherein the reactionconditions comprise an inert atmosphere (e.g., N₂, Ar, etc.).

In some embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out using a combination of any of themixture and reaction conditions disclosed above or elsewhere herein,e.g., in the Examples. Accordingly, in some embodiments, the methods ofthe present disclosure can be carried out wherein the reactionconditions comprise: (1) substrate loading of about 50-200 g/L compound(2); (2) engineered polypeptide concentration of about 1.5-2.5 g/L; (3)NADPH cofactor concentration of about 0.1-0.2 g/L; (4) a co-solventsolution of an aqueous buffer and about 60-70% (v/v) IPA; (5) about pH6.25-7.5; and (6) temperature of about 25-35° C.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise: (1) substrate loading of about 100 g/L compound (2); (2)engineered polypeptide concentration of about 2.0 g/L; (3) NADPHcofactor concentration of about 0.1 g/L; (4) a co-solvent solution of anaqueous buffer of 0.1M TEA and about 65% (v/v) IPA; (5) about pH 6.75;and (6) temperature of about 30 ° C.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise: (1) substrate loading of about 50-150 g/L of compound (2); (2)engineered polypeptide concentration of about 2.5-3.5 g/L; (3) NADPHcofactor concentration of about 0.1-0.2 g/L; (4) a co-solvent solutionof an aqueous buffer and about 15-25% (v/v) IPA and 25-35% (v/v)toluene; (5) about a pH of 6.6-7.0; and (6) a temperature of about28-30° C.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure can be carried out wherein the reaction conditionscomprise: (1) substrate loading of about 100 g/L of compound (2); (2)engineered polypeptide concentration of about 3.0 g/L; (3) NADPHcofactor concentration of about 0.1 g/L; (4) a co-solvent solution of anaqueous buffer of 0.1 M TEA and about 20% (v/v) IPA and 30% (v/v)toluene; (5) about a pH of 6.6-7.0; and (6) a temperature of about28-30° C.

Generally, in the methods disclosed herein, the biocatalytic reactionwith a polypeptide under suitable reaction conditions is allowed toproceed until essentially complete, or near complete, conversion ofcompound (2) to compound (1) is obtained. This conversion of substrateto product can be monitored using known methods by detecting substrateand/or product. Suitable methods include gas chromatography, HPLC, andthe like, and are described in the Examples.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure result in at least about 90% conversion of compound(2) at 100 g/L loading to compound (1) in 24 h, when carried out underreaction conditions of: engineered polypeptide concentration of about1.0-3.0 g/L; NADPH cofactor concentration of about 0.1 g/L; a co-solventsystem of at least 65% (v/v) IPA; and a temperature of 30° C. In someembodiments, the methods of the present disclosure when carried outunder these reaction conditions (e.g., 100 g/L compound (2) loading)result in at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater conversion of compound (2) to compound (1) in 24 h.

In some embodiments, the methods for preparing compound (1) of thepresent disclosure when carried out with 100 g/L compound (2) loadingresult in an diastereomeric excess of compound (1) of at least 97%, 98,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% in24 h.

In carrying out the conversion of compound (2) to compound (1) using theengineered polypeptides in the methods of the present disclosure, it isnecessary for an electron donor to be present. Generally, a cofactor isused is used as the electron donor in the reduction reaction. Thecofactor operates in combination with the polypeptides of the disclosurein the process. Suitable cofactors include, but are not limited to,NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH (the reducedform of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide) and NADH (thereduced form of NAD⁺). Generally, the reduced form of the cofactor isadded to the reaction mixture. Accordingly, in certain embodiments, themethods of the present disclosure are carried out wherein an electrondonor is present selected from NADPH cofactor or NADH cofactor. Incertain embodiments, the method can be carried out wherein the reactionconditions comprise an NADH or NADPH cofactor concentration of about0.03-0.5 g/L, about 0.05-0.3 g/L, about 0.1-0.2 g/L, about 0.5 g/L,about 0.1 g/L, or about 0.2 g/L.

The reduced NAD(P)H form can be optionally regenerated from the oxidizedNAD(P)⁺ form using a cofactor regeneration system. In some embodimentsof the process, a cofactor recycling system is used to regeneratecofactor NADPH/NADH form NADP⁺/NAD⁺ produced in the reaction.

In some embodiments of the process, an optional cofactor recyclingsystem can be used to regenerate cofactor NADPH/NADH form NADP+/NAD+produced in the reaction. A cofactor regeneration system refers to a setof reactants that participate in a reaction that reduces the oxidizedform of the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by thepolypeptide reduction of the keto substrate are regenerated in reducedform by the cofactor regeneration system. Cofactor regeneration systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst, that catalyzes the reductionof the oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and may be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

In some embodiments, the cofactor recycling system can comprise glucosedehydrogenase (GDH), which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Glucose dehydrogenases suitable for usein the practice of the processes described herein include both naturallyoccurring glucose dehydrogenases, as well as non-naturally occurringglucose dehydrogenases. Naturally occurring glucose dehydrogenaseencoding genes have been reported in the literature, e.g., the Bacillussubtilis 61297 GDH gene, B. cereus ATCC 14579 and B. megaterium.Non-naturally occurring glucose dehydrogenases generated using, forexample, mutagenesis, directed evolution, and the like and are providedin PCT publication WO 2005/018579, and US publication Nos. 2005/0095619and 2005/0153417. All of these sequences are incorporated herein byreference.

In some embodiments, the co-factor regenerating system can comprise aformate dehydrogenase, which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of formate and NAD⁺ or NADP⁺ to carbon dioxideand NADH or NADPH, respectively. Formate dehydrogenases that aresuitable for use as cofactor regenerating systems in the ketoreductasereactions described herein include naturally occurring and non-naturallyoccurring formate dehydrogenases. Suitable formate dehydrogenases aredescribed in PCT publication WO 2005/018579. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. A base or buffer may be usedto provide the desired pH.

In some embodiments, the co-factor regenerating system can comprise asecondary alcohol dehydrogenase, which is an NAD⁺ or NADP⁺-dependentenzyme that catalyzes the conversion of a secondary alcohol and NAD⁺ orNADP⁺ to a ketone and NADH or NADPH, respectively. Secondary alcoholdehydrogenases suitable for use as cofactor regenerating systems in theprocesses described herein include naturally occurring and non-naturallyoccurring ketoreductases. Naturally occurring secondary alcoholdehydrogenases include known alcohol dehydrogenases from,Thermoanerobium brockii, Rhodococcus erythropolis, Lactobacillus kefir,and Lactobacillus brevis, and non-naturally occurring secondary alcoholdehydrogenases include engineered alcohol dehydrogenases derivedtherefrom. In some embodiments, non-naturally occurring ketoreductasesengineered for thermo- and solvent stability can be used. Suchketoreductases are described in the present application and the patentpublications US 20080318295A1; US 20090093031A1; US 20090155863A1; US20090162909A1; US 20090191605A1; US 20100055751A1; WO/2010/025238A2;WO/2010/025287A2; and US 20100062499A1; each of which are incorporatedby reference herein.

The engineered ketoreductase polypeptides of the present disclosure haveimproved enzymatic activity for the conversion of IPA to acetonerelative to the naturally occurring ketoreductase polypeptide of SEQ IDNO: 2. Accordingly, in carrying out the conversion of compound (2) tocompound (1) using the engineered polypeptides in the methods of thepresent disclosure, the NADPH or NADH cofactor present can be recycledby the engineered polypeptide using IPA as reductant.

In certain embodiments, the methods comprising the conversion ofcompound (2) to compound (1) disclosed herein can be carried out withoutadding NADPH or NADH cofactor during the reaction and without any otherenzyme systems present (e.g., glucose dehydrogenase, or formatedehydrogenase).

In certain embodiments, the methods comprising the use of an engineeredpolypeptide of the present disclosure for the conversion of compound (2)to compound (1) can be carried out wherein no cofactor recycling enzymeis present other than the engineered polypeptide. For example, themethods comprising of the present disclosure can be carried out whereinthe reaction conditions comprise an IPA concentration is about 55-75%(v/v), an NADPH or NADH cofactor loading of about 0.03-0.5 g/L, andwherein no cofactor recycling enzyme is present other than theengineered polypeptide.

Suitable secondary alcohols useful in cofactor regenerating systemsinclude lower secondary alkanols and aryl-alkyl carbinols. Examples oflower secondary alcohols include isopropanol, 2-butanol,3-methyl-2-butanol, 2-pentanol, 3-pentanol, 3,3-dimethyl-2-butanol, andthe like. In one embodiment, the secondary alcohol is isopropanol.Suitable aryl-alkyl carbinols include unsubstituted and substituted1-arylethanols.

In some embodiments where the cofactor recycling system produces avolatile product, such as acetone from isopropanol, the volatile productcan be removed by sparging the reaction solution with a non-reactive gasor by applying a vacuum to lower the reaction pressure and removing thevolatile present in the gas phase. A non-reactive gas is any gas thatdoes not react with the reaction components. Various non-reactive gasesinclude nitrogen and noble gases (e.g., inert gases). In someembodiments, the non-reactive gas is nitrogen gas. For example, acetoneformed by oxidation of isopropanol can be removed by sparging withnitrogen gas or applying a vacuum to the reaction solution and removingthe acetone from the gas phase by an acetone trap, such as a condenseror other cold trap.

In the embodiments herein, the polypeptides carrying out the conversionof compound (2) to compound (1) and any additional enzymes of theoptional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the polypeptides disclosed hereinand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. Whole cellstransformed with gene(s) encoding the engineered ketoreductase enzymeand/or the optional cofactor regeneration enzymes, or cell extractsand/or lysates thereof, may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste).

Generally, the order of addition of reactants (e.g., substrate,cofactor, polypeptide) is not critical to the methods of the presentdisclosure. The reactants may be added together at the same time to asolvent (e.g., monophasic solvent, biphasic aqueous co-solvent system,and the like), or alternatively, some of the reactants may be addedseparately, and some together at different time points.

In some embodiments any of the above describe methods for the conversionof compound (2) to compound (1) can be carried out wherein the methodcomprises contacting an analog of compound (2) with an engineeredpolypeptide of the present disclosure (e.g., as described in Table 2 andelsewhere herein) in the presence of NADPH or NADH cofactor undersuitable reaction conditions, thereby resulting in the preparation ofthe chiral alcohol of the corresponding analog of compound (1) indiastereomeric excess. Suitable reactions conditions for the conversionof analogs of compound (2) to the chiral alcohol of the correspondinganalogs of compound (1) can be the same as used for compound (2) ordetermined by the ordinary artisan based on the known properties of theanalog compounds and routine experimentation.

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the analogs of compound (1) comprisingthe compound of Formula Ia shown below.

wherein,

X is selected from C or S;

R¹ is selected from —H, —F, —Cl, —Br, or —I;

R² is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle;

R³ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle;

R⁴ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group); and

R⁵ is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protectedwith a hydroxyl protecting group), —CH₂NH₂ (optionally protected with anitrogen protecting group), and any one of the following optionallysubstituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy,cycloalkyl, aryl, heteroaryl, or heterocycle.

Examples of hydroxyl protecting groups and nitrogen protecting groupsthat may be the R group of compounds of Formula IIa undergoing thebiocatalytic methods of the present disclosure can be found in P. G. M.Wuts and T. W. Greene, “Greene's Protective Groups in OrganicSynthesis—Fourth Edition,” John Wiley and Sons, New York, N.Y., 2007,Chapter 7 (“Greene”).

Accordingly, in some embodiments the present disclosure provides amethod of preparing a compound of Formula Ia comprising: contacting acompound of Formula IIa

wherein,

X, R¹, R², R³, R⁴, and R⁵, are defined as above for Formula Ia, with anengineered polypeptide of the present disclosure (e.g., as described inTable 2 and elsewhere herein) in the presence of NADPH or NADH cofactorunder suitable reaction conditions.

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which R² and R³ are eachindependently selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionallyprotected with a hydroxyl protecting group), —CH₂NH₂ (optionallyprotected with a nitrogen protecting group), and any one of thefollowing optionally substituted groups: —(C₁-C₆)alkyl, —(C₁-C₆)alkoxy,—(C₁-C₆)alkenyl, —(C₁-C₆)alkenoxy, —(C₁-C₆)alkynyl, —(C₁-C₆)alkynoxy,—(C₁-C₆)cycloalkyl, or a heterocycle, having from 1 to 4 carbon atomsand 1 to 2 hetero atoms.

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which R² and R³ are eachindependently selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionallyprotected with a hydroxyl protecting group), —CH₂NH₂ (optionallyprotected with a nitrogen protecting group), and any one of thefollowing optionally substituted groups: —(C₁-C₄)alkyl, —(C₁-C₄)alkoxy,—(C₁-C₄)alkenyl, —(C₁-C₄)alkenoxy, —(C₁-C₄)alkynyl, —(C₁-C₄)alkynoxy,—(C₁-C₄)cycloalkyl, or a heterocycle, having from 1 to 3 carbon atomsand 1 to 2 hetero atoms.

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which X is C, R² and R³ are eachindependently selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionallyprotected with a hydroxyl protecting group), and —CH₂NH₂ (optionallyprotected with a nitrogen protecting group).

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which X is C, R² and R³ are eachindependently selected from the following optionally substituted groups:—(C₁-C₄)alkyl, —(C₁-C₄)alkoxy, —(C₁-C₄)alkenyl, —(C₁-C₄)alkenoxy,—(C₁-C₄)alkynyl, and —(C₁-C₄)alkynoxy. In some embodiments, R² and R³are optionally substituted with one or more —OH groups.

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which X is C, any one or more ofR², R³, or R⁵ is an —OH or an —OH group protected with a hydroxylprotecting group. In some embodiments of the methods, the hydroxylprotecting group of R², R³, and/or R⁵ is selected from selected from thegroup consisting of benzyl, acetyl, benzoyl, tert-butyloxycarbonyl,silyl, tert-butyldiphenylsilyl, trimethylsilyl, para-methoxybenyl,benzylidine, dimethylacetal, and methoxy methyl. In some embodiments,the silyl group is —Si—(R^(a))(R^(b))(R^(c)) and R^(a), R^(b), and R^(c)are each independently selected from the group consisting of C₁-C₆alkyl, phenyl, acetyl, and benzyl groups.

In some embodiments, the present disclosure provides a method ofpreparing a compound of Formula Ia in which R¹ is —F, R² is —F, R⁴ is—H, and/or R⁵ is —H.

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the anti-hypercholesterolemic compoundsdescribed in PCT publication WO 2008/085300A1 based on compounds ofFormula Ib,

wherein,

R⁹ is selected from the group consisting of chloro, fluoro,—C≡C—(C₁-C₆)alkyl—NR¹⁰R¹¹, —(CH₂)_(x)CH═CH—(C₁-C₆)alkyl-NR¹⁰R¹¹,(C₁-C₈)alkyl-NR¹⁰R¹¹, —C≡C—(C₁-C₄)alkyl-CH—(CH₂—NR¹⁰R¹¹)₂,—(C₁-C₆)alkyl-CH—(CH₂—NR¹⁰R¹¹)₂, —C≡C—(C₁-C₆)alkyl-R^(11a),—(CH₂)_(x)CH═CH—(C₁-C₆)alkyl-R^(11a), —(C₁-C₈)alkyl-R^(11a),—C≡C—(C₁-C₆)alkyl, —(CH₂)_(x)CH═CH—(C₁-C₆)alkyl, —(C₁-C₈)alkyl,—(C₁-C₁₅)alkynyl mono- or poly-substituted with —OH and optionallysubstituted with R¹⁴, —(C₁-C₁₅)alkenyl mono- or poly-substituted with—OH and optionally substituted with R¹⁴, —(C₁-C₁₅)alkyl mono- orpoly-substituted with —OH and optionally substituted with R¹⁴ and x isan integer selected from 0, 1 and 2;

R¹⁰ is independently selected at each occurrence from the groupconsisting of —H and —(C₁-C₃)alkyl;

R¹¹ is independently selected at each occurrence from the groupconsisting of —H, —(C₁-C₃)alkyl, —C(O)—(C₁-C₃)alkyl, —C(O)—(C₁-C₃)alkyl,—C(O)—NR¹⁰R¹⁰, SO₂—(C₁-C₃)alkyl and —SO₂-phenyl;

R^(11a) is selected from the group consisting of —C(O)—NR¹⁰R¹⁰,—SO₂—(C₁-C₃)alkyl and —SO₂— phenyl;

R¹² is selected from the group consisting of —(C₂-C₁₅)alkynyl mono- orpoly-substituted with —OH and optionally substituted with R¹⁴,—(C₂-C₁₅)alkenyl mono- or poly-substituted with —OH and optionallysubstituted with R¹⁴, —(C₂-C₁₅)alkyl mono- or poly-substituted with —OHand optionally substituted with R¹⁴;

R¹³ is selected from the group consisting of —H and —OH; and

R¹⁴ is a sugar residue optionally substituted with —COOH,—COO—(C₁-C₃)alkyl and —(C₁-C₃)alkyl-OH; provided that when R⁹ isselected from the group consisting of —C≡C—(CH₂)₁₋₆—NR¹⁰R¹¹,—CH═CH—(CH₂)₁₋₆—NR¹⁰R¹¹ and —(CH₂)₁₋₈—NR¹⁰R¹¹, then R¹² is not selectedfrom the group consisting of —(C₁-C₁₅)alkyl mono- or poly-substitutedwith —OH, —CH═CH—(C₁-C₃)alkyl mono- or poly-substituted with —OH,—C≡C—(C₁-C₃)alkyl mono- or poly-substituted with —OH, and—(CH₂)₀₋₁—C(═CH₂)—CH₂OH.

Accordingly, in some embodiments the present disclosure provides amethod of preparing a compound of Formula Ib comprising: contacting acompound of Formula IIb,

wherein,

R⁹, R¹⁰, R¹¹, R^(11a), R¹², R¹³, and R¹⁴ are defined as above forFormula Ib, with an engineered polypeptide of the present disclosure(e.g., as described in Table 2 and elsewhere herein) in the presence ofNADPH or NADH cofactor under suitable reaction conditions.

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the anti-hypercholesterolemic compoundsdescribed in published Japan patent application 2010-83880, includingthe compound (1c) and other related analog compounds having substitutedpyridines or other heteroaryl at R⁹ of compound of Formula (Ib):

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the anti-hypercholesterolemic compoundsdescribed in PCT publication WO2010/056788, including the compound (1d)and other related analog compounds having substituted alkyl chains at R⁹and R¹² positions of the compound of Formula (Ib):

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the anti-hypercholesterolemic compoundsdescribed in US published patent application US2010/160282A1 and PCTPublication WO2010/100255.

In some embodiments, the analogs of compound (1) prepared using theabove described methods include the anti-hypercholesterolemic compoundsdescribed in US published patent application US2010/152156A1, includingthe compound of Formula (Ie) and other related analog compounds having asulfur atom at position X of the compound of Formula (Ia):

wherein:

R¹ is hydrogen, alkyl, halo, C₁₋₆ alkoxy or C₁₋₆ alkylS-;

R² is hydrogen, C₁₋₆ alkyl, halo or C₁₋₆ alkoxy;

R⁶ is hydrogen, C₁₋₆ alkyl, C₃₋₆ cycloalkyl or aryl;

R⁸, R⁹, R¹¹ and R¹² are independently hydrogen, a branched or unbranchedC₁₋₆ alkyl, C₃₋₆ cycloalkyl or aryl; wherein said C₁₋₆ alkyl may beoptionally substituted by one or more hydroxy, amino, guanidino, cyano,carbamoyl, carboxy, C₁₋₆alkoxy, aryl C₁₋₆alkoxy, (C₁₋₄alkyl)₃Si,N—(C₁₋₆alkyl)amino, N,N—(C₁₋₆alkyl)₂-amino, C₁₋₆alkylS(O)_(a),C₃₋₆cycloalkyl, aryl or aryl C₁₋₆ alkyl-S(O)_(a), wherein a is 0-2; andwherein any aryl group may be optionally substituted by one or twosubstituents selected from halo, hydroxy, C₁₋₆alkyl, C₁₋₆alkoxy, orcyano;

R⁷ and R¹⁰ is hydrogen, C₁₋₆ alkyl, or arylC₁₋₆ alkyl;

and, wherein R⁸ and R⁹ may form a ring with 2-7 carbon atoms and whereinR⁷ and R⁸ may form a ring with 3-6 carbon atoms; or a pharmaceuticallyacceptable salt, solvate, solvate of such a salt or a prodrug thereof.

6. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Example 1: Wild-Type Ketoreductase Gene Acquisition and Construction ofExpression Vectors

The wild-type ketoreductase gene from L. kefir (SEQ ID NO: 1) wasdesigned for expression in E. coli using standard codon optimization.(Codon-optimization software is reviewed in e.g., “OPTIMIZER: a webserver for optimizing the codon usage of DNA sequences,” Puigbò et al.,Nucleic Acids Res. 2007 July; 35(Web Server issue): W126-31. Epub 2007Apr 16.) Genes were synthesized using oligonucleotides composed of 42nucleotides and cloned into expression vector pCK110900 (vector depictedas FIG. 3 in US Patent Application Publication 20060195947, which ishereby incorporated by reference herein) under the control of a lacpromoter. The expression vector also contained the P15a origin ofreplication and the chloramphenicol resistance gene. Resulting plasmidswere transformed into E. coli W3110 (fhu-) using standard methods.Polynucleotides encoding the engineered ketoreductase polypeptides werealso cloned into vector pCK110900 for expression in E. coli W3110.

The engineered ketoreductase polypeptide of SEQ ID NO: 2 which wasderived previously based on directed evolution of a codon-optimized geneencoding the wild-type ketoreductase of Lactobacillus kefir (Genbankacc. No. AAP94029.1; GI: 33112056). SEQ ID NO: 2 has 19 amino acidresidue differences relative to the WT ketoreductase (D3N, G7S, L17Q,V95L, S96Q, G117S, Q127R, E145S, F147L, T152M, L153V, L176V, Y190C,D198K, L199D, E200P, K211R, I223V, and A241S). The polypeptide of SEQ IDNO: 2 was found to be able to convert compound (2) to compound (1)in >99% ee and with greater than 50% conversion rate in 20 h whileconverting IPA to acetone to recycle the NADP co-factor in 20% IPA(i.e., without a secondary enzyme for cofactor recycling) under initialscreening conditions (4 g/L compound (2) substrate; 0.5 g/L NADP, 100 mMTEA, pH 7.0, 1 mM MgSO₄, 25° C.). The polypeptide SEQ ID NO: 2 was usedas the starting backbone for subsequent rounds of evolution. Multiplerounds of directed evolution of the gene encoding SEQ ID NO: 2 (i.e.,SEQ ID NO: 1) were carried out. Each round used the gene encoding themost improved engineered polypeptide from each round as the parent“backbone” sequence for the subsequent round of evolution. The resultingengineered ketoreductase polypeptide sequences and specific mutationsand relative activities are listed in Table 2.

Example 2: Production of Engineered Ketoreductase Polypeptides

The engineered ketoreductase polypeptides of the disclosure wereproduced in E. coli W3110 as an intracellular protein expressed underthe control of the lac promoter. The polypeptide accumulates primarilyas a soluble cytosolic active enzyme. A shake-flask procedure is used togenerate engineered polypeptide powders that can be used in activityassays or biocatalytic process disclosed herein.

Fermentation for shake flask powders: A single microbial colony of E.coli containing a plasmid encoding an engineered ketoreductase ofinterest is inoculated into 50 mL Luria Bertani broth containing 30μg/ml chloramphenicol and 1% glucose. Cells are grown overnight (atleast 16 hours) in an incubator at 30° C. with shaking at 250 rpm. Theculture is diluted into 250 mL Terrific Broth (12 g/L bacto-tryptone, 24g/L yeast extract, 4 mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1mM MgSO₄) containing 30 μg/ml chloramphenicol, in a 1 liter flask to anoptical density at 600 nm (OD600) of 0.2 and allowed to grow at 30° C.Expression of the ketoreductase gene is induced by addition ofisopropyl-β-D-thiogalactoside (“IPTG”) to a final concentration of 1 mMwhen the OD600 of the culture is 0.6 to 0.8 and incubation is thencontinued overnight (at least 16 hours). Cells are harvested bycentrifugation (5000 rpm, 15 min, 4° C.) and the supernatant discarded.

Production of ketoreductase shake-flask powders: The cell pellet isresuspended with an equal volume of cold (4° C.) 100 mM triethanolamine(chloride) buffer, pH 7.0 (optionally including 2 mM MgSO₄), andharvested by centrifugation as above. The washed cells are resuspendedin two volumes of the cold triethanolamine (chloride) buffer and passedthrough a French Press twice at 12,000 psi while maintained at 4° C.Cell debris is removed by centrifugation (9000 rpm, 45 minutes, 4° C.).The clear lysate supernatant was collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry shake-flask powderof crude ketoreductase polypeptide. Alternatively, the cell pellet(before or after washing) can be stored at 4° C. or −80° C.

Fermentation for production downstream process (DSP) powders:Larger-scale (˜100-120 g) fermentation of the engineered ketoreductasesfor production of DSP powders can be carried out as a short batchfollowed by a fed batch process according to standard bioprocessmethods. Briefly, ketoreductase expression is induced by addition ofIPTG to a final concentration of 1 mM. Following fermentation, the cellsare harvested and resuspended in 100 mM Triethanolamine-H₂SO₄ buffer,then mechanically disrupted by homogenization. The cell debris andnucleic acid are flocculated with polyethylenimine (PEI) and thesuspension clarified by centrifugation. The resulting clear supernatantis concentrated using a tangential cross-flow ultrafiltration membraneto remove salts and water. The concentrated and partially purifiedenzyme concentrate can then be dried in a lyophilizer and packaged(e.g., in polyethylene containers).

Example 3: Activity Assay of Engineered Ketoreductase Polypeptides

High-throughput growth & expression: Picked and grown using standardKRED protocol for W3110 with direct induction: (1) Master growth=singlecolonies picked from agar Q-trays by Q-bot and grown overnight in LBmedia containing 1% glucose and 30 μg/mL CAM, 30° C., 200 rpm, 85%humidity. (2) Subculture=20 μL of overnight growth transferred to a deepwell plate containing 380 μL 2xYT growth media containing 30 μg/mL CAM,1 mM IPTG, 1 mM MgSO₄, and incubated for ˜18 h at 30° C., 200 rpm, 85%humidity. (3) Cell culture centrifuged at 4000 rpm, 4° C. for 10 min.,and used media discarded. Cell pellets resuspended in between 200-400 μLlysis buffer (100 mM TEA buffer, pH7.0, containing 1 mM MgSO₄, 400 μg/mLPMBS and 500 μg/mL Lysozyme.

High Throughput Screening Procedure: 60-140 μL of a 5.7-100 g/L solutionof the substrate in either a mixture of toluene:IPA:acetone (v/v/v ratioof 5:9:1-15:9:1) or IPA:acetone (v/v ratio of 49:1) was added to eachwell of a Costar™ deep-well 96-well plate. Subsequently, 40-120 μL of a0.25-1.25 g/L solution of NADP in 100 mM TEA buffer, pH 7.0 containing 1mM MgSO₄ was also added. Finally, 20 μL of a freshly prepared suspensionof lysed cells in lysis buffer (either concentrated or diluted up to20-fold in 100 mM TEA buffer pH 7.0 containing 1 mM MgSO₄) was added tomake the total volume in each well 200 μL ([substrate]=4-80 g/L,[NADP]=0.1-0.5 g/L, Solvent=either toluene:IPA:acetone:buffer (relative% volumes of 10:18:2:70 or 30:18:2:50) or IPA:acetone:buffer (relative %volumes of 65:35 or 70:30). The plate was then heat sealed and shakenfor 2 or 24 h at RT, 30° C., or 37° C. before 0.8 mL of acetonitrile wasadded to each well to quench the reaction.

The specific conditions of the High Throughput Screening procedure canbe varied in order to identify variant polypeptides having amino aciddifferences providing different improved properties relative to theselected reference polypeptide. Typically, the stringency of screeningconditions are increased through the course of the directed evolution ofthe variant polypeptides. Conditions that can be varied includesubstrate concentration, cofactor concentration, solvent conditions,temperature, and total reaction time. Exemplary modifications of thescreening conditions are noted in Table 2.

Analytical method used for activity assay: The plate containing thereactions quenched with acetonitrile were heat sealed, and shaken for 5minutes, prior to being centrifuged at 4,000 rpm for 10 m. 200 μL of thesupernatant was then transferred to a Costar™ round bottom 96-well plateand heat sealed prior to HPLC analysis. HPLC was performed using a C-18Symmetry 100×4.6 mm, 5 μm column, with isocratic elution of a 66%MeCN:34% H₂O solvent mixture at a flow of 2.5 mL/min. Both substrate andproduct were detected by UV absorbance at 254 nm.

Example 4: Biocatalytic Process I for Preparation of Compound (1)(Ezetimibe) from Substrate Compound (2)

This example illustrates a first biocatalytic process using anengineered ketoreductase polypeptide of the disclosure to prepareEzetimibe (compound (1)) on a 10 g scale. The biocatalytic reaction iscarried out in an aqueous co-solvent system of TEA buffer (100 mM, pH7), 30% toluene, 20% IPA, and a substrate loading is 100 g/L. Theengineered ketoreductase (polypeptide of SEQ ID NO: 80 at 3 g/L loading)uses the cofactor NADPH (0.1 g/L loading) as a reducing agent, which isoxidized to NADP⁺ during the reaction. The engineered ketoreductase alsoacts as the secondary alcohol dehydrogenase in an in-situ “recyclingsystem” to regenerate the reduced form of the cofactor through theoxidation of the IPA co-solvent to acetone. The product in thebiocatalytic reaction is extracted into THF and solvent swap withtoluene provides the desired crude product of compound (1), which isthen further crystallized from THF/toluene.

Preparation of compound (2) substrate: Compound (2) for use as substratein the biocatalytic reaction can be prepared by oxidation of samples ofthe Ezetimibe API (compound (1)) according to the following procedure.An oven dried 2-neck 500 mL RB flask equipped with a thermocouple, amagnetic stir bar, and a nitrogen gas inlet was charged with the whitepowder of compound (1) (32.06 g, 78.3 mmol) and N-methyl morpholineoxide (NMO) (18.3 g, 156.6 mmol). 300 mL of anhydrous dichloromethanewas added, affording a clear yellow solution. Oven dried activated 4 Åmolecular sieves (35 g) was added, and the solution was cooled to 8° C.(internal temperature) using an ice/NaCl bath. Tetrapropylammoniumperruthenate (TPAP) (2.75 g, 7.83 mmol) was added in one portion to theflask. The internal temperature rose to 15.4° C. then slowly dropped to8° C. The ice bath was removed, and the reaction mixture was allowed tostir at 25° C. for 2.5 h. The dark brown solution was filtered through a4″ bed of Celite and rinsed with dichloromethane (1.5 L) and diethylether (500 mL). The filtrate was monitored by TLC to ensure all ketoneproduct had been eluted. The sample was concentrated under reducedpressure and purified by column chromatography with 25% EtOAc in heptaneyielding compound (2) as an off-white solid, 21.01 g (68% Th), 100% pureat UV₂₅₄. HPLC analysis of a compound (2) sample in acetonitrile (˜1-5mg/mL) can be run on an Eclipse XDB-C18 column under the followingconditions: T=35° C.; mobile phase A=water+0.1% TFA; mobile phaseB=acetonitrile+0.1% TFA; run time=10 m; 0-4.5 min=25% to 90% B; 4.5-5.25min=90% B; 5.25-6 min=90%-25% B; post-time=1 min at 25% B. UV detectionat 214 and 254 nm. Compound (2) retention time=5.85 m.

Biocatalytic reaction procedure: A 250 mL round bottomed flask wasequipped with overhead stirrer and internal thermometer. The reactor wascharged sequentially with 300 mg DSP powder of engineered ketoreductasepolypeptide of SEQ ID NO: 80, 50.0 mL 100 mM TEA buffer (pH 7), 10.0 mgNADP dissolved in buffer, 10.0 g of compound (2) dissolved in 30 mLtoluene, and 20 mL IPA. The resulting slurry reaction was heated to 30°C. (internal temperature), stirring at ˜500 rpm. The final temperaturewas reached within 15 min. The reaction was run at a starting pH of 7and the pH remained constant throughout the reaction time. The reactioncourse was followed periodically by taking samples out of the reactionmixture, quenching, and analyzing as described in HPLC Method 1. At 24h, the reaction solution was a white suspension and the Method 1in-process analysis indicated 82% conversion. At 48 h, in-processanalysis indicated 94% conversion, and the reaction was cooled to 25° C.

Crude product work-up procedure: THF (60 mL) was charged to the reactionmixture at room temperature and agitated at 250 rpm for 15 minutes.Phases were allowed to separate and the aqueous layer removed. The THFphase was collected separately. The purity of the product in the THFphase was determined to 94.6% according to HPLC (Method 1). Toluene (60mL) was added, and the resulting solution was concentrated toapproximately 60 mL on rotary evaporator at 40° C. and incrementallyreducing the pressure to 70 Torr. Toluene (60 mL) was added again, andthe resulting hazy solution was concentrated to approx 90 mL on rotaryevaporator at 40° C. and incrementally reducing the pressure to 70 Torr.At this stage product precipitated as white solid and GC analysis of theorganic (THF) layer indicated that ≤2.0% THF remained. The precipitatedproduct was recovered by filtration and the residue was washed with 1×15mL of toluene and dried under vacuum (approx 20 mm Hg) for 24 hours.This provided: 8.75 g (90% yield) chiral alcohol of compound (1) as awhite solid; chemical purity of 97.2% (AUC, HPLC Method 1).

Crystallization procedure: To a suspension of 8.0 g crude compound (1)in toluene (80 mL) at 82° C. (internal), THF (10 mL) was added slowlywhile stirring. The slurry became clear solution at the end of theaddition. The solution was allowed to cool to room temperature (25° C.)over a period of 12 hours while stirring magnetically (100 RPM). Theresulting white precipitate was filtered under reduced pressure. Thewhite residue was washed once with cold toluene (10 mL) and dried undervacuum (˜20 mm Hg) for 24 hours. This provided: 6.3 g (68% yield) ofcompound (1) in a single crop as a white solid; chemical purity=99.9%(AUC, HPLC Method 1); chiral purity >99.9% d.e. (Method 2).

Analytical methods using in the process of Example 4: Samples wereanalyzed for percent conversion and/or diastereomeric purity using HPLCaccording to Method 1 or Method 2 as described below. HPLC samples wereprepared as follows: 10 μL are taken from the reaction suspension viapipette, dissolved in 1 mL of acetonitrile, and injected neat into theHPLC according the Method 1 or Method 2 parameters.

HPLC Method 1 parameters for monitoring biocatalytic reaction progressare shown in Table 4.

TABLE 4 Method 1 HPLC parameters Instrument Agilent HPLC 1200 seriesColumn Symmetry C18 4.6 × 100 mm Mobile Phase 70% Acetonitrile + 0.1%TFA, 30% Water + 0.1% TFA isocratic Flow Rate 1.0 ml/min DetectionWavelength 280.0 nm Detector Temperature 45° C. Injection Volume 10 μlRun time 4.0 min Retention times Product [Compound (1)]: 2.03 minSubstrate [Compound (2)]: 2.60 min Toluene: 3.67 min Response factor 1.4(Substrate/Product)

Method 2 HPLC parameters for determining diastereomeric purity ofbiocatalytic reaction product are shown in Table 5.

TABLE 5 Method 2 HPLC parameters Instrument Agilent HPLC 1200 seriesColumn Chiralpak AD-H 4.6 × 250 mm (5 um) Mobile Phase 80% Heptane/20%EtOH (0-18 min, isocratic) 50% Heptane/50% EtOH (18.5-33 min, isocratic)Flow Rate 1.0 ml/min Detection Wavelength 230.0 nm Detector Temperature20° C. Injection Volume 10 μl Run time 45.0 min Retention timesSubstrate [Compound (2)]: 31.56 min Product [Compound (1)]: 16.44 min(R, R, S) diastereomer: 15.05 min

Example 5: Process II for Preparation of Ezetimibe from Compound (2)

This example illustrates a second biocatalytic process using anengineered ketoreductase polypeptide of the disclosure to prepareEzetimibe (Compound (1)) on a 20 g scale.

Biocatalytic reaction procedure: A 500 mL jacketed reactor was chargedsequentially with the following: 20 g compound (2) (assayed at 88% w/wpurity) as solid, 130 mL IPA, 55 mL TEA buffer, 2.0 mL of TEA buffersolution containing 20 mg NADP⁺, 13.0 mL of TEA buffer solutioncontaining 400 mg of the engineered KRED of SEQ ID NO: 168. Theresulting reaction mixture was stirred at 30° C. (internal) at ˜250 rpm.The pH of the reaction mixture ranges between 6.30-6.40 at 30° C., withan initial buffer pH of 6.75 at RT. The reaction course was followedperiodically by taking samples from the reaction mixture, quenching, andanalyzing as described in Method 3. Percent conversion at 4 h, 18 h, and19 h, was 72.0%, 98.2%, and 98.4%, respectively. After in-processanalysis (Method 3) indicated maximum possible conversion (at 98.4%conversion) the reaction mixture was taken for the subsequent workup andisolation procedure.

Product work-up and isolation: Acetone formed during the reaction wasdistilled under vacuum (40 torr) at 30° C. To the thick slurry, water(200 mL) was added and the distillation continued at a slightly elevatedtemperature (40 torr, 40° C.) until about 25% IPA remained relative tothe start of distillation. The slurry with the crude product was drainedfrom the reactor. The reactor was washed with another 200 mL of waterand drained into the same container from above. The crude product wascollected by filtration through a sintered funnel and washed with 50 mLwater. The wet cake was dried for 15 h under vacuum (5 torr) at 25° C.Upon drying, 16.0 g of crude product was obtained 99.0% chemical purity(AUC, HPLC, 99.8% d.e.). Yield of crude product is 90% with respect tothe effective loading of keto phenol substrate (17.6 g). The crudeproduct was further purified by recrystallization as described below.

Recrystallization: A suspension of crude product (10.0 g) in IPA (30.0mL) was heated to 60° C. (internal) to allow maximum dissolution ofproduct. The hot solution from above was passed through a celite (5.0 g)bed in a sintered funnel. Upon complete filtration, the celite bed waswashed with pre-heated IPA (30.0 mL, ˜60° C.). Distillation to drynessof the combined filtrates from above showed >9.0 g of product. The whitesolid was stirred in 30.0 mL IPA and heated to 60° C. (internal) toobtain a clear solution. Water (40.0 mL) was added drop wise to theabove solution at 60° C. and the resultant solution was allowed to coolto 25° C. The crystallized product was filtered through a sinteredfunnel and dried under vacuum (5 torr, 25° C.) for 15 h. This provided:8.8 g of chiral alcohol product of compound (1) in a single crop as awhite solid; 99.5% chemical purity (AUC, HPLC, 99.9% d.e.). Essentiallythe only detectable impurity was the keto phenol substrate, measuring0.38% (AUC, HPLC).

Analytical methods used in the process of Example 5: an HPLC method fordetermination of % conversion (Method 3); and a chiral HPLC method fordetermination of diastereomeric purity (Method 4).

Method 3 sample preparation for HPLC: 0.3 mL of reaction mixture wassampled from the stirred reaction suspension via pipette. The appearanceof the sample should be as finely dispersed as the reaction mixtureitself. The sample was fully dissolved in 25 mL of methanol oracetonitrile. Injection is neat into the HPLC.

The HPLC parameters used for determination of percent conversionaccording to Method 3 are shown in Table 6.

TABLE 6 Instrument Varian 920-LC series Column Alltima C18, 53 × 7 mm, 3μm with guard column (P/N: 50605) Mobile Phase 60% Acetonitrile, 40%Water (Isocratic) Flow Rate 1.3 mL/min Detection Wavelength 254.0 nmColumn Temperature Ambient Injection Volume 10 μL Run time 5.0 minRetention times Product Compound (1): 2.67 min Keto phenol SubstrateCompound (2): 3.92 min Response Factor 1.46 (Substrate/Product)

Method 4 HPLC sample preparation of in-process sample: 10 μL of reactionmixture is sampled from the stirred reaction suspension via micropipetteand added to 1 mL of absolute ethanol in an HPLC glass vial ready foranalysis.

Method 4 HPLC sample preparation of final product sample: 1mL ofabsolute ethanol is added directly to 1 mg of sample in an HPLC glassvial. Ensure full dissolution before submitting to HPLC for analysis.

The HPLC parameters used for determination of percent diastereomericpurity of product according to Method 4 are shown in Table 7.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A non-naturally occurring ketoreductasepolypeptide having ketoreductase activity, comprising an amino acidsequence that has at least 80% sequence identity to SEQ ID NO: 168,wherein the residue at position 40 is arginine.
 2. The non-naturallyoccurring ketoreductase polypeptide of claim 1, wherein the amino acidsequence of said non-naturally occurring ketoreductase polypeptidefurther comprises the following feature: the residue at position 190 isalanine or proline.
 3. The non-naturally occurring ketoreductasepolypeptide of claim 1, wherein the amino acid sequence of saidnon-naturally occurring ketoreductase polypeptide further comprises oneor more of the features selected from: the residue at position X93 is Aand the residue at position X94 is T; the residue at position X93 is Aand the residue at position X94 is P; the residue at position X93 is Aand the residue at position X94 is S; or the residue at position X93 isI and the residue at position X94 is S.
 4. The non-naturally occurringketoreductase of claim 1, wherein the amino acid sequence of saidnon-naturally occurring ketoreductase polypeptide further comprises oneor more of the features selected from: the residue at position X203 isG; the residue at position X202 is G and the residue at position X203 isG; or the residue at position X201 is A, the residue position X202 is Gand the residue at position X203 is G.
 5. The non-naturally occurringketoreductase claim 1, wherein the amino acid sequence of saidnon-naturally occurring ketoreductase polypeptide further comprises oneor more of the features selected from: the residue at position X196 isT; and the residue at position X199 is F.
 6. The non-naturally occurringketoreductase of claim 1, wherein the amino acid sequence of saidnon-naturally occurring ketoreductase polypeptide further comprises oneor more of the features selected from: the residue at position X21 is Ror F; the residue at position X25 is R, T, or N; the residue at positionX64 is V; the residue at position X93 is A; the residue at position X94is T, S, or P; the residue at position X95 is M, or V; the residue atposition X96 is V, G, A, N, S, P, or T; the residue at position X99 isL; the residue at position X108 is H or K; the residue at position X117is G; the residue at position X127 is K, or Q; the residue at positionX147 is M, or I; the residue at position X148 is I; the residue atposition X150 is H; the residue at position X152 is F, or N; the residueat position X155 is C; the residue at position X163 is I; the residue atposition X195 is M; the residue at position X201 is I, L, or A; theresidue at position X202 is L, N, V, or G; the residue at position X203is G; the residue at position X204 is V, or A; the residue at positionX205 is V; the residue at position X206 is I; the residue at positionX207 is T, C, I, or N; the residue at position X211 is K; the residue atposition X221 is D; the residue at position X223 is I; or the residue atposition X226 is V.
 7. A non-naturally occurring polynucleotide encodingthe non-naturally occurring ketoreductase of claim
 1. 8. An expressionvector comprising the polynucleotide of claim
 7. 9. The expressionvector of claim 7, further comprising at least one control sequence. 10.A host cell comprising the non-naturally occurring polynucleotide ofclaim
 7. 11. A host cell comprising the expression vector of claim 8.12. A method for preparing a non-naturally occurring ketoreductasepolypeptide comprising culturing a host cell of claim 11, underconditions such that said non-naturally occurring ketoreductasepolypeptide is produced and isolating the non-naturally occurringpolypeptide from the cell.